Nucleic acid-associated proteins

ABSTRACT

A Various embodiments of the invention provide human nucleic acid-associated proteins (NAAP) and polynucleotides which identify and encode NAAP. Embodiments of the invention also provide expression vectors, host cells, antibodies, agonists, and antagonists. Other embodiments provide methods for diagnosing, treating, or preventing disorders associated with aberrant expression of NAAP.

TECHNICAL FIELD

The invention relates to novel nucleic acids, nucleic acid-associatedproteins encoded by these nucleic acids, and to the use of these nucleicacids and proteins in the diagnosis, treatment, and prevention of cellproliferative, neurological, developmental, and autoimmune/inflammatorydisorders, and infections. The invention also relates to the assessmentof the effects of exogenous compounds on the expression of nucleic acidsand nucleic acid-associated proteins.

BACKGROUND OF THE INVENTION

Multicellular organisms are comprised of diverse cell types that differdramatically both in structure and function. The identity of a cell isdetermined by its characteristic pattern of gene expression, anddifferent cell types express overlapping but distinctive sets of genesthroughout development. Spatial and temporal regulation of geneexpression is critical for the control of cell proliferation, celldifferentiation, apoptosis, and other processes that contribute toorganismal development. Furthermore, gene expression is regulated inresponse to extracellular signals that mediate cell-cell communicationand coordinate the activities of different cell types. Appropriate generegulation also ensures that cells function efficiently by expressingonly those genes whose functions are required at a given time.

Transcription Factors

Transcriptional regulatory proteins are essential for the control ofgene expression. Some of these proteins function as transcriptionfactors that initiate, activate, repress, or terminate genetranscription. Transcription factors generally bind to the promoter,enhancer, and upstream regulatory regions of a gene in asequence-specific manner, although some factors bind regulatory elementswithin or downstream of a gene coding region. Transcription factors maybind to a specific region of DNA singly or as a complex with otheraccessory factors. (Reviewed in Lewin, B. (1990) Genes IV, OxfordUniversity Press, New York, N.Y., and Cell Press, Cambridge, Mass., pp.554-570.)

The double helix structure and repeated sequences of DNA createtopological and chemical features which can be recognized bytranscription factors. These features are hydrogen bond donor andacceptor groups, hydrophobic patches, major and minor grooves, andregular, repeated stretches of sequence which induce distinct bends inthe helix. Typically, transcription factors recognize specific DNAsequence motifs of about 20 nucleotides in length. Multiple, adjacenttranscription factor-binding motifs may be required for gene regulation.

Many transcription factors incorporate DNA-binding structural motifswhich comprise either α helices or β sheets that bind to the majorgroove of DNA. Four well-characterized structural motifs arehelix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix.Proteins containing these motifs may act alone as monomers, or they mayform homo- or heterodimers that interact with DNA.

The helix-turn-helix motif consists of two α helices connected at afixed angle by a short chain of amino acids. One of the helices binds tothe major groove. Helix-turn-helix motifs are exemplified by thehomeobox motif which is present in homeodomain proteins. These proteinsare critical for specifying the anterior-posterior body axis duringdevelopment and are conserved throughout the animal kingdom. TheAntennapedia and Ultrabithorax proteins of Drosophila melanogaster areprototypical homeodomain proteins. (Pabo, C. O. and R. T. Sauer (1992)Annu. Rev. Biochem. 61:1053-1095.)

Mouse HES-6 is a member of the Hairy/Enhancer-of-split (HES) family ofbasic helix-loop-helix transcription factors. HES genes act as nucleareffectors of Notch signaling to regulate the transcriptional activity ofseveral Notch target genes. HES-6 is expressed in all neurogenicplacodes and their derivatives and in the brain, where it is patternedalong both the anteroposterior and dorsoventral axes. HES-6 is alsoexpressed in embryonic tissues where Notch signaling controls cell-fatedecisions, such as the trunk, the dorsal root ganglia, myotomes, andthymus. In the limb buds HES-6 is expressed in skeletal muscle andpresumptive tendons. It is also expressed in epithelial cells of theembryonic respiratory, urinary and digestive systems (Vasiliauskas, D.and Stern C. D. (2000) Mech. Dev. 98:133-137; Pissarra, L. et al. (2000)Mech Dev 95:275-278).

The zinc finger motif, which binds zinc ions, generally contains tandemrepeats of about 30 amino acids consisting of periodically spacedcysteine and histidine residues. Examples of this sequence pattern,designated C2H2 and C3HC4 (“RING” finger), have been described. (Lewin,supra.) Zinc finger proteins each contain an α helix and an antiparallelβ sheet whose proximity and conformation are maintained by the zinc ion.Contact with DNA is made by the arginine preceding the α helix and bythe second, third, and sixth residues of the α helix. Variants of thezinc finger motif include poorly defined cysteine-rich motifs which bindzinc or other metal ions. These motifs may not contain histidineresidues and are generally nonrepetitive. The zinc finger motif may berepeated in a tandem array within a protein, such that the α helix ofeach zinc finger in the protein makes contact with the major groove ofthe DNA double helix. This repeated contact between the protein and theDNA produces a strong and specific DNA-protein interaction. The strengthand specificity of the interaction can be regulated by the number ofzinc finger motifs within the protein. Though originally identified inDNA-binding proteins as regions that interact directly with DNA, zincfingers occur in a variety of proteins that do not bind DNA (Lodish, H.et al. (1995) Molecular Cell Biology, Scientific American Books, NewYork, N.Y., pp. 447-451). For example, Galcheva-Gargova, Z. et al.(1996) Science 272:1797-1802) have identified zinc finger proteins thatinteract with various cytokine receptors.

The C2H2-type zinc finger signature motif contains a 28 amino acidsequence, including 2 conserved Cys and 2 conserved His residues in aC-2-C-12-H-3-H type motif. The motif generally occurs in multiple tandemrepeats. A cysteine-rich domain including the motif Asp-His-His-Cys(DHHC-CRD) has been identified as a distinct subgroup of zinc fingerproteins. The DHHC-CRD region has been implicated in growth anddevelopment. One DHHC-CRD mutant shows defective function of Ras, asmall membrane-associated GTP-binding protein that regulates cell growthand differentiation, while other DHHC-CRD proteins probably function inpathways not involving Ras (Bartels, D. J. et al. (1999) Mol. Cell Biol.19:6775-6787).

Zinc-finger transcription factors are often accompanied by modularsequence motifs such as the Kruppel-associated box (KRAB) and the SCANdomain. For example, the hypoalphalipoproteinemia susceptibility geneZNF202 encodes a SCAN box and a KRAB domain followed by eight C2H2zinc-finger motifs (Honer, C. et al. (2001) Biochim. Biophys. Acta1517:441-448). The SCAN domain is a highly conserved, leucine-rich motifof approximately 60 amino acids found at the amino-terminal end of zincfinger transcription factors. SCAN domains are most often linked to C2H2zinc finger motifs through their carboxyl-terminal end. Biochemicalbinding studies have established the SCAN domain as a selective hetero-and homotypic oligomerization domain. SCAN domain-mediated proteincomplexes may function to modulate the biological function oftranscription factors (Schumacher, C. et al. (2000) J. Biol. Chem.275:17173-17179).

The KRAB (Kruppel-associated box) domain is a conserved amino acidsequence spanning approximately 75 amino acids and is found in almostone-third of the 300 to 700 genes encoding C2H2 zinc fingers. The KRABdomain is found N-terminally with respect to the finger repeats. TheKRAB domain is generally encoded by two exons; the KRAB-A region or boxis encoded by one exon and the KRAB-B region or box is encoded by asecond exon. The function of the KRAB domain is the repression oftranscription. Transcription repression is accomplished by recruitmentof either the KRAB-associated protein-1, a transcriptional corepressor,or the KRAB-A interacting protein. Proteins containing the KRAB domainare likely to play a regulatory role during development (Williams, A. J.et al. (1999) Mol. Cell Biol. 19:8526-8535). A subgroup of highlyrelated human KRAB zinc finger proteins detectable in all human tissuesis highly expressed in human T lymphoid cells (Bellefroid, E. J. et al.(1993) EMBO J. 12:1363-1374). The ZNF85 KRAB zinc finger gene, a memberof the human ZNF91 family, is highly expressed in normal adult testis,in serninomas, and in the NT2/D1 teratocarcinoma cell line (Poncelet, D.A. et al. (1998) DNA Cell Biol.17:931-943).

The Kruppel protein regulates Drosophila segmentation. There areapproximately 300 genes which encode such proteins in the whole humangenome. In fact, more than 100 different mRNAs encoding Kruppelmultifingered proteins, most of them novel, have been found in the humanplacenta. The sequences of the 106 finger repeats present in nine ofthese proteins are highly homologous. There are a few positions locatedin the alpha-helical structure which show variability. Research impliesthat this variability impacts the DNA-binding specificity of theproteins (Bellefroid, E. J. et al. (1989) DNA 8:377-387).

ZNF143 is a human zinc finger Kruppel family protein of the GLI type. Itis 84% identical to the Xenopus laevis selenocysteine tRNA genetranscription activating factor (Staf). Staf is implicated in theenhanced transcription of small nuclear RNA (snRNA) and snRNA-type genesby RNA polymerases II (Pol II) and III (Pol III). Staf also possessesthe capacity to stimulate expression from a Pol II mRNA promoter.ZNF143, along with the related ZNF138 and ZNF139, is localized tochromosome regions 7q11.2, 7q21.3-q22.1, and 11p15.3-p15.4. Theseregions are involved in deletion and/or translocations associated withWilliams syndrome, split hand and foot disease (SHFD1), andBeckwith-Wiedemann syndrome, respectively, suggesting that ZNF143 geneis involved in developmental and malignant disorders. ZNF143 mRNAs areexpressed in many normal adult tissues, including leukocytes, colon,small intestine, ovary, testis, prostate, thymus, and spleen tissues.Further, transcription of the mouse chaperone-encoding Ccta gene isregulated by ZNF143 and another Staf family zinc-finger transcriptionfactor, ZNF76, implying that these RNA and chaperone genes arecoregulated to facilitate synthesis of mature proteins during activecell growth (Tommerup, N. and Vissing, H. (1995) Genomics 27: 259-264;Myslinski, E. et al. (1998) J. Biol. Chem. 273:21998-2006; Kubota, H. etal. (2000) J. Biol. Chem. 275:28641-28648).

The C4 motif is found in hormone-regulated proteins. The C4 motifgenerally includes only 2 repeats. A number of eukaryotic and viralproteins contain a conserved cysteine-rich domain of 40 to 60 residues(called C3HC4 zinc-finger or RING finger) that binds two atoms of zinc,and is probably involved in mediating protein-protein interactions. The3D “cross-brace” structure of the zinc ligation system is unique to theRING domain. The spacing of the cysteines in such a domain isC-x(2)-C-x(9 to 39)-C-x(1 to 3)-H-x(2 to 3)-C-x(2)-C-x(4 to48)-C-x(2)-C. The PHD finger is a C4HC3 zinc-finger-like motif found innuclear proteins thought to be involved in chromatin-mediatedtranscriptional regulation.

GATA-type transcription factors contain one or two zinc finger domainswhich bind specifically to a region of DNA that contains the consecutivenucleotide sequence GATA. NMR studies indicate that the zinc fingercomprises two irregular anti-parallel β sheets and an α helix, followedby a long loop to the C-terminal end of the finger (Ominchinski, J. G.(1993) Science 261:438-446). The helix and the loop connecting the twoβ-sheets contact the major groove of the DNA, while the C-terminal part,which determines the specificity of binding, wraps around into the minorgroove.

The LIM motif consists of about 60 amino acid residues and containsseven conserved cysteine residues and a histidine within a consensussequence (Schmeichel, K. L. and Beckerle, M. C. (1994) Cell 79:211-219).The LIM family includes transcription factors and cytoskeletal proteinswhich may be involved in development, differentiation, and cell growth.One example is actin-binding LIM protein, which may play roles inregulation of the cytoskeleton and cellular morphogenesis (Roof, D. J.et al. (1997) J. Cell Biol. 138:575-588). The N-terminal domain ofactin-binding LIM protein has four double zinc finger motifs with theLIM consensus sequence. The C-terminal domain of actin-binding LIMprotein shows sequence similarity to known actin-binding proteins suchas dematin and villin. Actin-binding LIM protein binds to F-actinthrough its dematin-like C-terminal domain. The LIM domain may mediateprotein-protein interactions with other LIM-binding proteins.

Myeloid cell development is controlled by tissue-specific transcriptionfactors. Myeloid zinc finger proteins (MZF) include MZF-1 and MZF-2.MZF-1 functions in regulation of the development of neutrophilicgranulocytes. A murine homolog MZF-2 is expressed in myeloid cells,particularly in the cells committed to the neutrophilic lineage. MZF-2is down-regulated by G-CSF and appears to have a unique function inneutrophil development (Murai, K et al. (1997) Genes Cells 2:581-591).

The leucine zipper motif comprises a stretch of amino acids rich inleucine which can form an anphipathic α helix. This structure providesthe basis for dimerization of two leucine zipper proteins. The regionadjacent to the leucine zipper is usually basic, and upon proteindimerization, is optimally positioned for binding to the major groove.Proteins containing such motifs are generally referred to as bZIPtranscription factors. The leucine zipper motif is found in theproto-oncogenes Fos and Jun, which comprise the heterodimerictranscription factor AP1 involved in cell growth and the determinationof cell lineage (Papavassiliou, A. G. (1995) N. Engl. J. Med.332:45-47).

The mouse kreisler (kr) mutation causes segmentation abnormalities inthe caudal hindbrain and defective inner ear development. The kr cDNAencodes a basic domain-leucine zipper (bZIP) transcription factor inwhich a serine is substituted for an asparagine residue conserved in theDNA-binding domain of all known bZIP family members. The identity,expression, and mutant phenotype of kr indicate an early role in axialpatterning and provide insights into the molecular and embryologicmechanisms that govern hindbrain and otic development (Cordes, S. P. andBarsh, G. S. (1994) Cell 79:1025-1034).

The helix-loop-helix motif (HLH) consists of a short α helix connectedby a loop to a longer α helix. The loop is flexible and allows the twohelices to fold back against each other and to bind to DNA. Thetranscription factor Myc contains a prototypical HLH motif.

The NF-kappa-B/Rel signature defines a family of eukaryotictranscription factors involved in oncogenesis, embryonic development,differentiation and immune response. Most transcription factorscontaining the Rel homology domain (RHD) bind as dimers to a consensusDNA sequence motif termed kappa-B. Members of the Rel family share ahighly conserved 300 amino acid domain termed the Rel homology domain.The characteristic Rel C-terminal domain is involved in gene activationand cytoplasmic anchoring functions. Proteins known to contain the RHDdomain include vertebrate nuclear factor NF-kappa-B, which is aheterodimer of a DNA-binding subunit and the transcription factor p65,mammalian transcription factor ReIB, and vertebrate proto-oncogenec-rel, a protein associated with differentiation and lymphopoiesis(Kabrun, N. and Enrietto, P. J. (1994) Semin. Cancer Biol. 5:103-112).

A DNA binding motif termed ARID (AT-rich interactive domain)distinguishes an evolutionarily conserved family of proteins. Theapproximately 100-residue ARID sequence is present in a series ofproteins strongly implicated in the regulation of cell growth,development, and tissue-specific gene expression. ARID proteins includeBright (a regulator of B-cell-specific gene expression), dead ringer(involved in development), and MRF-2 (which represses expression fromthe cytomegalovirus enhancer) (Dallas, P. B. et al. (2000) Mol. CellBiol. 20:3137-3146).

The ELM2 (Egl-27 and MTA1 homology 2) domain is found inmetastasis-associated protein MTA1 and protein ER1. The Caenorhabditiselegans gene egl-27 is required for embryonic patterning MTA1, a humangene with elevated expression in metastatic carcinomas, is a componentof a protein complex with histone deacetylase and nucleosome remodellingactivities (Solari, F. et al. (1999) Development 126:2483-2494). TheELM2 domain is usually found to the N terminus of a myb-like DNA bindingdomain. ELM2 is also found associated with an ARID DNA.

LEF-1 is a transcription factor that participates in the regulation ofthe T-cell receptor alpha (TCR alpha) enhancer by facilitating theassembly of multiple proteins into a higher order nucleoprotein complex.The function of LEF-1 is dependent, in part, on the HMG domain. Thisdomain induces a sharp bend in the DNA helix and on an activation domainthat stimulates transcription only in a specific context of otherenhancer-binding proteins. ALY is a LEF-1-interacting protein which is aubiquitously expressed, nuclear protein that specifically associateswith the activation domains of LEF-1 and AML-1 (acute myeloid leukemia1). AML-1 is another protein component of the TCR alpha enhancercomplex. ALY increases DNA binding by both LEF-1 and AML proteins.Overexpression of ALY stimulates the activity of the TCR alpha enhancercomplex in transfected nonlymphoid HeLa cells, whereas down-regulationof ALY by anti-sense oligonucleotides eliminates TCR alpha enhanceractivity in T cells. Similar to LEF-1, ALY can stimulate transcriptionin the context of the TCR alpha enhancer but apparently not whentethered to DNA through an heterologous DNA-binding domain. Researchsuggests that ALY mediates context-dependent transcriptional activationby facilitating the functional collaboration of multiple proteins in theTCR alpha enhancer complex (Bruhn, L. et al. (1997) Genes Dev.11:640-653).

A family of nuclear proteins, designated SL3-3 enhancer factors 2(SEF2), interact with an Ephrussi box-like motif within theglucocorticoid response element in the enhancer of the murine leukemiavirus SL3-3. Mutation of the DNA sequence decreased the basal enhanceractivity in various cell lines. The important nucleotides for binding ofSEF2 are conserved in most type C retroviruses. Various cell typesdisplayed differences both in the sets of SEF2-DNA complexes formed andin their amounts. A cDNA which encoded a protein, SEF2-1A, thatinteracted specifically with the SEF2-binding sequence has been isolatedfrom human thymocytes. The nucleotide sequence specificity of therecombinant SER2-1A, expressed in Escherichia coli, corresponds to thatof one of the nuclear SEF2 proteins (Corneliussen, B. et al. J(1991) J.Virol. 65:6084-6093).

The Iroquois (Irx) family of genes are found in nematodes, insects andvertebrates. Irx genes usually occur in one or two genomic clusters ofthree genes each and encode transcriptional controllers that possess acharacteristic homeodomain. The Irx genes function early in developmentto specify the identity of diverse territories of the body. Later indevelopment in both Drosophila and vertebrates, the Irx genes functionagain to subdivide those territories into smaller domains. (For a reviewof Iroquois genes, see Cavodeassi, F. et al. (2001) Development128:2847-2855.) For example, mouse and human Irx4 proteins are 83%conserved and their 63-aa homeodomain is more than 93% identical to thatof the Drosophila Iroquois patterning genes. lrx4 transcripts arepredominantly expressed in the cardiac ventricles. The homeobox geneIrx4 mediates ventricular differentiation during cardiac development(Bruneau, B. G. et al. (2000) Dev. Biol. 217:266-77).

Histidine triad (HIT) proteins share residues in distinctive dimeric,10-stranded half-barrel structures that form two identical purinenucleotide-binding sites. Hint (histidine triad nucleotide-bindingprotein)-related proteins, found in all forms of life, and fragilehistidine triad (Fhit)-related proteins, found in animals and fungi,represent the two main branches of the HIT superfamily. Fhit homologsbind and cleave diadenosine polyphosphates. Fhit-Ap(n)A complexes appearto function in a proapoptotic tumor suppression pathway in epithelialtissues (Brenner C. et al. (1999) J. Cell Physiol.181:179-187).

Most transcription factors contain characteristic DNA binding motifs,and variations on the above motifs and new motifs have been and arecurrently being characterized. (Faisst, S. and S. Meyer (1992) NucleicAcids Res. 20:3-26.)

Chromatin Associated Proteins

In the nucleus, DNA is packaged into chromatin, the compact organizationof which limits the accessibility of DNA to transcription factors andplays a key role in gene regulation. (Lewin, supra, pp. 409-410.) Thecompact structure of chromatin is determined and influenced bychromatin-associated proteins such as the histones, the high mobilitygroup (HMG) proteins, and the chromodomain proteins. There are fiveclasses of histones, H1, H2A, H2B, H3, and H4, all of which are highlybasic, low molecular weight proteins. The fundamental unit of chromatin,the nucleosome, consists of 200 base pairs of DNA associated with twocopies each of H2A, H2B, H3, and H4. H1 links adjacent nucleosomes. HMGproteins are low molecular weight, non-histone proteins that may IS playa role in unwinding DNA and stabilizing single-stranded DNA.Chromodomain proteins play a key role in the formation of highlycompacted heterochromatin, which is transcriptionally silent.

The SWI/SNF complex in yeast facilitates the function of transcriptionalactivators by opposing chromatin-dependent repression of transcription.In mammals SWI/SNF complexes are present in multiple forms made up of9-12 proteins known as BRG1-associated factors (BAFs) ranging from 47 to250 kD. BRG1-associated factors (BAFs) include the SWI2-SNF2 homologwhich interacts with and activates human immunodeficiency virusintegrase and is homologous to the yeast SNF5 gene (Wang, W. et al.(1996) Genes Dev. 10:2117-2130).

Diseases and Disorders Related to Gene Regulation

Many neoplastic disorders in humans can be attributed to inappropriategene expression. Malignant cell growth may result from either excessiveexpression of tumor promoting genes or insufficient expression of tumorsuppressor genes. (Cleary, M. L. (1992) Cancer Surv. 15:89-104.) Thezinc finger-type transcriptional regulator WT1 is a tumor-suppressorprotein that is inactivated in children with Wilm's tumor. The oncogenebc1-6, which plays an important role in large-cell lymphoma, is also azinc-finger protein (Papavassiliou, A. G. (1995) N. Engl. J. Med.332:45-47). Chromosomal translocations may also produce chimeric locithat fuse the coding sequence of one gene with the regulatory regions ofa second unrelated gene. Such an arrangement likely results ininappropriate gene transcription, potentially contributing tomalignancy. In Burkitt's lymphoma, for example, the transcription factorMyc is translocated to the immunoglobulin heavy chain locus, greatlyenhancing Myc expression and resulting in rapid cell growth leading toleukemia (Latchman, D. S. (1996) N. Engl. J. Med. 334:28-33).

In addition, the immune system responds to infection or trauma byactivating a cascade of events that coordinate the progressiveselection, amplification, and mobilization of cellular defensemechanisms. A complex and balanced program of gene activation andrepression is involved in this process. However, hyperactivity of theimmune system as a result of improper or insufficient regulation of geneexpression may result in considerable tissue or organ damage. Thisdamage is well-documented in immunological responses associated witharthritis, allergens, heart attack, stroke, and infections. (Isselbacheret al. Harrison's Principles of Internal Medicine, 13/e, McGraw Hill,Inc. and Teton Data Systems Software, 1996.) The causative gene forautoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED)was recently isolated and found to encode a protein with two PHD-typezinc finger motifs (Bjorses, P. et al. (1998) Hum. Mol. Genet.7:1547-1553).

Furthermore, the generation of multicellular organisms is based upon theinduction and coordination of cell differentiation at the appropriatestages of development. Central to this process is differential geneexpression, which confers the distinct identities of cells and tissuesthroughout the body. Failure to regulate gene expression duringdevelopment could result in developmental disorders. Human developmentaldisorders caused by mutations in zinc finger-type transcriptionalregulators include: urogenital developmental abnormalities associatedwith WT1; Greig cephalopolysyndactyly, Pallister-Hall syndrome, andpostaxial polydactyly type A (GL13), and Townes-Brocks syndrome,characterized by anal, renal, limb, and ear abnormalities (SALL1)(Engelkamp, D. and V. van Heyningen (1996) Curr. Opin. Genet. Dev.6:334-342; Kohlhase, J. et al. (1999) Am. J. Hum. Genet. 64:435-445).

Human acute leukemias involve reciprocal chromosome translocations thatfuse the ALL-1 gene located at chromosome region 11q23 to a series ofpartner genes positioned on a variety of human chromosomes. The fusedgenes encode chimeric proteins. The AF17 gene encodes a protein of 1093amino acids, containing a leucine-zipper dimerization motif located 3′of the fusion point and a cysteine-rich domain at the N terminus thatshows homology to a domain within the protein Br140 (peregrin) (PrasadR. et al. (1994) Proc. Natl. Acad. Sci. U S A 91:8107-8111).

Synthesis of Nucleic Acids

Polymerases

DNA and RNA replication are critical processes for cell replication andfunction. DNA and RNA replication are mediated by the enzymes DNA andRNA polymerase, respectively, by a “templating” process in which thenucleotide sequence of a DNA or RNA strand is copied by complementarybase-pairing into a complementary nucleic acid sequence of either DNA orRNA. However, there are fundamental differences between the twoprocesses.

DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotideto the 3′-OH end of a polynucleotide strand (the primer strand) that ispaired to a second (template) strand. The new DNA strand therefore growsin the 5′ to 3′ direction (Alberts, B. et al. (1994) The MolecularBiology of the Cell, Garland Publishing Inc., New York, N.Y., pp251-254). The substrates for the polymerization reaction are thecorresponding deoxynucleotide triphosphates which must base-pair withthe correct nucleotide on the template strand in order to be recognizedby the polymerase. Because DNA exists as a double-stranded helix, eachof the two strands may serve as a template for the formation of a newcomplementary strand. Each of the two daughter cells of a dividing celltherefore inherits a new DNA double helix containing one old and one newstrand. Thus, DNA is said to be replicated “semiconservatively” by DNApolymerase. In addition to the synthesis of new DNA, DNA polymerase isalso involved in the repair of damaged DNA as discussed below under“Ligases.”

In contrast to DNA polymerase, RNA polymerase uses a DNA template strandto “transcribe” DNA into RNA using ribonucleotide triphosphates assubstrates. Like DNA polymerization, RNA polymerization proceeds in a 5′to 3′ direction by addition of a ribonucleoside monophosphate to the3′-OH end of a growing RNA chain. DNA transcription generates messengerRNAs (mRNA) that carry information for protein synthesis, as well as thetransfer, ribosomal, and other RNAs that have structural or catalyticfunctions. In eukaryotes, three discrete RNA polymerases synthesize thethree different types of RNA (Alberts, supra, pp. 367-368). RNApolymerase I makes the large ribosomal RNAs, RNA polymnerase II makesthe mRNAs that will be translated into proteins, and RNA polymerase IIImakes a variety of small, stable RNAs, including 5S ribosomal RNA andthe transfer RNAs (tRNA). In all cases, RNA synthesis is initiated bybinding of the RNA polymerase to a promoter region on the DNA andsynthesis begins at a start site within the promoter. Synthesis iscompleted at a stop (termination) signal in the DNA whereupon both thepolymerase and the completed RNA chain are released.

Ligases

DNA repair is the process by which accidental base changes, such asthose produced by oxidative damage, hydrolytic attack, or uncontrolledmethylation of DNA, are corrected before replication or transcription ofthe DNA can occur. Because of the efficiency of the DNA repair process,fewer than one in a thousand accidental base changes causes a mutation(Alberts, supra, pp. 245-249). The three steps common to most types ofDNA repair are (1) excision of the damaged or altered base or nucleotideby DNA nucleases, (2) insertion of the correct nucleotide in the gapleft by the excised nucleotide by DNA polymerase using the complementarystrand as the template and, (3) sealing the break left between theinserted nucleotide(s) and the existing DNA strand by DNA ligase. In thelast reaction, DNA ligase uses the energy from ATP hydrolysis toactivate the 5′ end of the broken phosphodiester bond before forming thenew bond with the 3′-OH of the DNA strand. In Bloom's syndrome, aninherited human disease, individuals are partially deficient in DNAligation and consequently have an increased incidence of cancer(Alberts, supra p. 247).

Nucleases

Nucleases comprise enzymes that hydrolyze both DNA (DNase) and RNA(Rnase). They serve different purposes in nucleic acid metabolism.Nucleases hydrolyze the phosphodiester bonds between adjacentnucleotides either at internal positions (endonucleases) or at theterminal 3′ or 5′ nucleotide positions (exonucleases). A DNA exonucleaseactivity in DNA polymerase, for example, serves to remove improperlypaired nucleotides attached to the 3′-OH end of the growing DNA strandby the polymerase and thereby serves a “proofreading” function. Asmentioned above, DNA endonuclease activity is involved in the excisionstep of the DNA repair process.

RNases also serve a variety of functions. For example, RNase P is aribonucleoprotein enzyme which cleaves the 5′ end of pre-tRNAs as partof their maturation process. RNase H digests the RNA strand of anRNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, andRNase H is an important enzyme in the retroviral replication cycle.Pancreatic RNase secreted by the pancreas into the intestine hydrolyzesRNA present in ingested foods. RNase activity in serum and cell extractsis elevated in a variety of cancers and infectious diseases (Schein, C.H. (1997) Nat. Biotechnol. 15:529-536). Regulation of RNase activity isbeing investigated as a means to control tumor angiogenesis, allergicreactions, viral infection and replication, and fungal infections.

Modification of Nucleic Acids

Methylases

Methylation of specific nucleotides occurs in both DNA and RNA, andserves different functions in the two macromolecules. Methylation ofcytosine residues to form 5-methyl cytosine in DNA occurs specificallyin CG sequences which are base-paired with one another in the DNAdouble-helix. The pattern of methylation is passed from generation togeneration during DNA replication by an enzyme called “maintenancemethylase” that acts preferentially on those CG sequences that arebase-paired with a CG sequence that is already methylated. Suchmethylation appears to distinguish active from inactive genes bypreventing the binding of regulatory proteins that “turn on” the gene,but permiting the binding of proteins that inactivate the gene (Alberts,supra pp. 448-451). In RNA metabolism, “tRNA methylase” produces one ofseveral nucleotide modifications in tRNA that affect the conformationand base-pairing of the molecule and facilitate the recognition of theappropriate mRNA codons by specific tRNAs. The primary methylationpattern is the dimethylation of guanine residues to form N,N-dimethylguanine.

Helicases and Single-Stranded Binding Proteins

Helicases are enzymes that destabilize and unwind double helixstructures in both DNA and RNA. Since DNA replication occurs more orless simultaneously on both strands, the two strands must first separateto generate a replication “fork” for DNA polymerase to act on. Two typesof replication proteins contribute to this process, DNA helicases andsingle-stranded binding proteins. DNA helicases hydrolyze ATP and usethe energy of hydrolysis to separate the DNA strands. Single-strandedbinding proteins (SSBs) then bind to the exposed DNA strands, withoutcovering the bases, thereby temporarily stabilizing them for templatingby the DNA polymerase (Alberts, supra, pp. 255-256).

RNA helicases also alter and regulate RNA conformation and secondarystructure. Like the DNA helicases, RNA helicases utilize energy derivedfrom ATP hydrolysis to destabilize and unwind RNA duplexes. The mostwell-characterized and ubiquitous family of RNA helicases is theDEAD-box family, so named for the conserved B-type ATP-binding motifwhich is diagnostic of proteins in this family. Over 40 DEAD-boxhelicases have been identified in organisms as diverse as bacteria,insects, yeast, amphibians, mammals, and plants. DEAD-box helicasesfunction in diverse processes such as translation initiation, splicing,ribosome assembly, and RNA editing, transport, and stability. Examplesof these RNA helicases include yeast Drs1 protein, which is involved inribosomal RNA processing; yeast TIF1 and TIF2 and mammalian eIF-4A,which are essential to the initiation of RNA translation; and human p68antigen, which regulates cell growth and division (Ripmaster, T. L. etal. (1992) Proc. Natl. Acad. Sci. USA 89:11131-11135; Chang, T.-H. etal. (1990) Proc. Natl. Acad. Sci. USA 87:1571-1575). These RNA helicasesdemonstrate strong sequence homology over a stretch of some 420 aminoacids. Included among these conserved sequences are the consensussequence for the A motif of an ATP binding protein; the “DEAD box”sequence, associated with ATPase activity; the sequence SAT, associatedwith the actual helicase unwinding region; and an octapeptide consensussequence, required for RNA binding and ATP hydrolysis (Pause, A. et al.(1993) Mol. Cell Biol. 13:6789-6798). Differences outside of theseconserved regions are believed to reflect differences in the functionalroles of individual proteins (Chang, T. H. et al. (1990) Proc. Natl.Acad. Sci. USA 87:1571-1575).

Some DEAD-box helicases play tissue- and stage-specific roles inspermatogenesis and embryogenesis. Overexpression of the DEAD-box 1protein (DDX1) may play a role in the progression of neuroblastoma (Nb)and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem.273:21161-21168). These observations suggest that DDX1 may promote orenhance tumor progression by altering the normal secondary structure andexpression levels of RNA in cancer cells. Other DEAD-box helicases havebeen implicated either directly or indirectly in tumorigenesis.(Discussed in Godbout, supra.) For example, murine p68 is mutated inultraviolet light-induced tumors, and human DDX6 is located at achromosomal breakpoint associated with B-cell lymphoma. Similarly, achimeric protein comprised of DDX10 and NUP98, a nucleoporin protein,may be involved in the pathogenesis of certain myeloid malignancies.

Topoisomerases

Besides the need to separate DNA strands prior to replication, the twostrands must be “unwound” from one another prior to their separation byDNA helicases. This function is performed by proteins known as DNAtopoisomerases. DNA topoisomerase effectively acts as a reversiblenuclease that hydrolyzes a phosphodiesterase bond in a DNA strand,permits the two strands to rotate freely about one another to remove thestrain of the helix, and then rejoins the original phosphodiester bondbetween the two strands. Topoisomerases are essential enzymesresponsible for the topological rearrangement of DNA brought about bytranscription, replication, chromatin formation, recombination, andchromosome segregation. Superhelical coils are introduced into DNA bythe passage of processive enzymes such as RNA polymerase, or by theseparation of DNA strands by a helicase prior to replication. Knottingand concatenation can occur in the process of DNA synthesis, storage,and repair. All topoisomerases work by breaking a phosphodiester bond inthe ribose-phosphate backbone of DNA. A catalytic tyrosine residue onthe enzyme makes a nucleophilic attack on the scissile phosphodiesterbond, resulting in a reaction intermediate in which a covalent bond isformed between the enzyme and one end of the broken strand. Atyrosine-DNA phosphodiesterase functions in DNA repair by hydrolyzingthis bond in occasional dead-end topoisomerase I-DNA intermediates(Pouliot, J. J. et al. (1999) Science 286:552-555).

Two types of DNA topoisomerase exist, types I and II. Type Itopoisomerases work as monomers, making a break in a single strand ofDNA while type II topoisomerases, working as homodimers, cleave bothstrands. DNA Topoisomerase I causes a single-strand break in a DNA helixto allow the rotation of the two strands of the helix about theremaining phosphodiester bond in the opposite strand. DNA topoisomeraseI[ causes a transient break in both strands of a DNA helix where twodouble helices cross over one another. This type of topoisomerase canefficiently separate two interlocked DNA circles (Alberts, supra,pp.260-262). Type II topoisomerases are largely confined toproliferating cells in eukaryotes, such as cancer cells. For this reasonthey are targets for anticancer drugs. Topoisomerase II has beenimplicated in multi-drug resistance (MDR) as it appears to aid in therepair of DNA damage inflicted by DNA binding agents such as doxorubicinand vincristine.

The topoisomerase I family includes topoisomerases I and III (topo I andtopo III). The crystal structure of human topoisomerase I suggests thatrotation about the intact DNA strand is partially controlled by theenzyme. In this “controlled rotation” model, protein-DNA interactionslimit the rotation, which is driven by torsional strain in the DNA(Stewart, L. et al. (1998) Science 379:1534-1541). Structurally, topo Ican be recognized by its catalytic tyrosine residue and a number ofother conserved residues in the active site region. Topo I is thought tofunction during transcription. Two topo IIIs are known in humans, andthey are homologous to prokaryotic topoisomerase I, with a conservedtyrosine and active site signature specific to this family. Topo III hasbeen suggested to play a role in meiotic recombination. A mouse topo IIIis highly expressed in testis tissue and its expression increases withthe increase in the number of cells in pachytene (Seki, T. et al. (1998)J. Biol. Chem. 273:28553-28556).

The topoisomerase II family includes two isozymes (IIα and IIβ) encodedby different genes. Topo II cleaves double stranded DNA in areproducible, nonrandom fashion, preferentially in an AT rich region,but the basis of cleavage site selectivity is not known. Structurally,topo II is made up of four domains, the first two of which arestructurally similar and probably distantly homologous to similardomains in eukaryotic topo I. The second domain bears the catalytictyrosine, as well as a highly conserved pentapeptide. The Ha isoformappears to be responsible for unlinkig DNA during chromosomesegregation. Cell lines expressing IIα but not IIβ suggest that IIIβ isdispensable in cellular processes; however, IIβ knockout mice diedperinatally due to a failure in neural development. That the majorabnormalities occurred in predominandy late developmental events(neurogenesis) suggests that IIβ is needed not at mitosis, but ratherduring DNA repair (Yang, X. et al. (2000) Science 287:131-134).

Topoisomerases have been implicated in a number of disease states, andtopoisomerase poisons have proven to be effective anti-tumor drugs forsome human malignancies. Topo I is mislocalized in Fanconi's anemia, andmay be involved in the chromosomal breakage seen in this disorder(Wunder, E. (1984) Hum. Genet. 68:276-281). Overexpression of atruncated topo III in ataxia-telangiectasia (A-T) cells partiallysuppresses the A-T phenotype, probably through a dominant negativemechanism. This suggests that topo III is deregulated in A-T (Fritz, E.et al. (1997) Proc. Natl. Acad. Sci. USA 94:4538-4542). Topo III alsointeracts with the Bloom's Syndrome gene product, and has been suggestedto have a role as a tumor suppressor (Wu, L. et al. (2000) J. Biol.Chem. 275:9636-9644). Aberrant topo II activity is often associated withcancer or increased cancer risk. Greatly lowered topo II activity hasbeen found in some, but not all A-T cell lines (Mohamed, R. et al.(1987) Biochem. Biophys. Res. Commun. 149:233-238). On the other hand,topo II can break DNA in the region of the A-T gene (ATM), whichcontrols all DNA damage-responsive cell cycle checkpoints (Kaufmann, W.K. (1998) Proc. Soc. Exp. Biol. Med. 217:327-334). The ability oftopoisomerases to break DNA has been used as the basis of antitumordrugs. Topoisomerase poisons act by increasing the number of dead-endcovalent DNA-enzyme complexes in the cell, ultimately triggering celldeath pathways (Fortune, J. M. and N. Osheroff (2000) Prog. Nucleic AcidRes. Mol. Biol. 64:221-253; Guichard, S. M. and M. K. Danks (1999) Curr.Opin. Oncol. 11:482-489). Antibodies against topo I are found in theserum of systemic sclerosis patients, and the levels of the antibody maybe used as a marker of pulmonary involvement in the disease (Diot, E. etal. (1999) Chest 116:715-720). Finally, the DNA binding region of humantopo I has been used as a DNA delivery vehicle for gene therapy (Chen,T. Y. et al. (2000) Appl. Microbiol. Biotechnol 53:558-567).

Recombinases

Genetic recombination is the process of rearranging DNA sequences withinan organism's genome to provide genetic variation for the organism inresponse to changes in the environment. DNA recombination allowsvariation in the particular combination of genes present in anindividual's genome, as well as the timing and level of expression ofthese genes. (See Alberts, supra pp. 263-273.) Two broad classes ofgenetic recombination are commonly recognized, general recombination andsite-specific recombination. General recombination involves geneticexchange between any homologous pair of DNA sequences usually located ontwo copies of the same chromosome. The process is aided by enzymes,recombinases, that “nick” one strand of a DNA duplex more or lessrandomly and permit exchange with a complementary strand on anotherduplex. The process does not normally change the arrangement of genes ina chromosome. In site-specific recombination, the recombinase recognizesspecific nucleotide sequences present in one or both of the recombiningmolecules. Base-pairing is not involved in this form of recombinationand therefore it does not require DNA homology between the recombiningmolecules. Unlike general recombination, this form of recombination canalter the relative positions of nucleotide sequences in chromosomes.

RNA Metabolism

Ribonucleic acid (RNA) is a linear single-stranded polymer of fournucleotides, ATP, CTP, UTP, and GTP. In most organisms, RNA istranscribed as a copy of deoxyribonucleic acid (DNA), the geneticmaterial of the organism. In retroviruses RNA rather than DNA serves asthe genetic material. RNA copies of the genetic material encode proteinsor serve various structural, catalytic, or regulatory roles inorganisms. RNA is classified according to its cellular localization andfunction. Messenger RNAs (mRNAs) encode polypeptides. Ribosomal RNAs(rRNAs) are assembled, along with ribosomal proteins, into ribosomes,which are cytoplasmic particles that translate mRNA into polypeptides.Transfer RNAs (tRNAs) are cytosolic adaptor molecules that function inmRNA translation by recognizing both an mRNA codon and the amino acidthat matches that codon. Heterogeneous nuclear RNAs (hnRNAs) includemRNA precursors and other nuclear RNAs of various sizes. Small nuclearRNAs (snRNAs) are a part of the nuclear spliceosome complex that removesintervening, non-coding sequences (introns) and rejoins exons inpre-mRNAs.

Proteins are associated with RNA during its transcription from DNA, RNAprocessing, and translation of mRNA into protein. Proteins are alsoassociated with RNA as it is used for structural, catalytic, andregulatory purposes.

RNA Processing

Ribosomal RNAs (rRNAs) are assembled, along with ribosomal proteins,into ribosomes, which are cytoplasmic particles that translate messengerRNA (mRNA) into polypeptides. The eukaryotic ribosome is composed of a60S (large) subunit and a 40S (small) subunit, which together form the80S ribosome. In addition to the 18S, 28S, 5S, and 5.8S rRNAs, ribosomescontain from 50 to over 80 different ribosomal proteins, depending onthe organism. Ribosomal proteins are classified according to whichsubunit they belong (i.e., L, if associated with the large 60S largesubunit or S if associated with the small 40S subunit). E. coliribosomes have been the most thoroughly studied and contain 50 proteins,many of which are conserved in all life forms. The structures of nineribosomal proteins have been solved to less than 3.0 D resolution (i.e.,S5, S6, S17, L1, L6, L9, L12, L14, L30), revealing common motifs, suchas b-a-b protein folds in addition to acidic and basic RNA-bindingmotifs positioned between b-strands. Most ribosomal proteins arebelieved to contact rRNA directly (reviewed in Liljas, A. and Garber, M.(1995) Curr. Opin. Struct. Biol. 5:721-727; see also Woodson, S. A. andLeontis, N. B. (1998) Curr. Opin. Struct. Biol. 8:294-300; Ramakrishnan,V. and White, S. W. (1998) Trends Biochem. Sci. 23:208-212).

Ribosomal proteins may undergo post-translational modifications orinteract with other ribosome-associated proteins to regulatetranslation. For example, the highly homologous 40S ribosomal protein S6kinases (S6K1 and S6K2) play a key role in the regulation of cell growthby controlling the biosynthesis of translational components which makeup the protein synthetic apparatus (including the ribosomal proteins).In the case of S6K1, at least eight phosphorylation sites are believedto mediate kinase activation in a hierarchical fashion (Dufner andThomas (1999) Exp. Cell. Res. 253:100-109). Some of the ribosomalproteins, including L1, also function as translational repressors bybinding to polycistronic mRNAs encoding ribosomal proteins (reviewed inLiljas, supra and Garber, supra).

Recent evidence suggests that a number of ribosomal proteins havesecondary functions independent of their involvement in proteinbiosynthesis. These proteins function as regulators of cellproliferation and, in some instances, as inducers of cell death. Forexample, the expression of human ribosomal protein L13a has been shownto induce apoptosis by arresting cell growth in the G2/M phase of thecell cycle. Inhibition of expression of L13a induces apoptosis in targetcells, which suggests that this protein is necessary, in the appropriateamount, for cell survival. Similar results have been obtained in yeastwhere inactivation of yeast homologues of L13a, rp22 and rp23, resultsin severe growth retardation and death. A closely related ribosomalprotein, L7, arrests cells in G1 and also induces apoptosis. Thus, itappears that a subset of ribosomal proteins may function as cell cyclecheckpoints and compose a new family of cell proliferation regulators.

Mapping of individual ribosomal proteins on the surface of intactribosomes is accomplished using 3D immunocryoelectronmicroscopy, wherebyantibodies raised against specific ribosomal proteins are visualized.Progress has been made toward the mapping of L1, L7, and L12 while thestructure of the intact ribosome has been solved to only 20-25Dresolution and inconsistencies exist among different crude structures(Frank, J. (1997) Curr. Opin. Struct. Biol. 7:266-272).

Three distinct sites have been identified on the ribosome. Theaminoacyl-tRNA acceptor site (A site) receives charged tRNAs (with theexception of the initiator-tRNA). The peptidyl-tRNA site (P site) bindsthe nascent polypeptide as the amino acid from the A site is added tothe elongating chain. Deacylated tRNAs bind in the exit site (E site)prior to their release from the ribosome. The structure of the ribosomeis reviewed in Stryer, L. (1995) Biochemistr, W. H. Freeman and Company,New York N.Y., pp. 888-9081; Lodish, H. et al. (1995) Molecular CellBiology, Scientific American Books, New York N.Y., pp. 119-138; andLewin, B (1997) Genes VI, Oxford University Press, Inc. New York, N.Y.).

Various proteins are necessary for processing of transcribed RNAs in thenucleus. Pre-mRNA processing steps include capping at the 5′ end withmethylguanosine, polyadenylating the 3′ end, and splicing to removeintrons. The primary RNA transcript from DNA is a faithful copy of thegene containing both exon and intron sequences, and the latter sequencesmust be cut out of the RNA transcript to produce a mRNA that codes for aprotein. This “splicing” of the mRNA sequence takes place in the nucleuswith the aid of a large, multicomponent ribonucleoprotein complex knownas a spliceosome. The spliceosomal complex is comprised of five smallnuclear ribonucleoprotein particles (snRNPs) designated U1, U2, U4, U5,and U6. Each snRNP contains a single species of snRNA and about tenproteins. The RNA components of some snRNPs recognize and base-pair withintron consensus sequences. The protein components mediate spliceosomeassembly and the splicing reaction. Autoantibodies to snRNP proteins arefound in the blood of patients with systemic lupus erythematosus(Stryer, L. (1995) Biochemistry, W. H. Freeman and Company, New YorkN.Y., p. 863).

Heterogeneous nuclear ribonucleoproteins nRNPs) have been identifiedthat have roles in splicing, exporting of the mature RNAs to thecytoplasm, and mRNA translation (Biamonti, G. et al. (1998) Clin. Exp.Rheumatol. 16:317-326). Some examples of hnRNPs include the yeastproteins Hrp1p, involved in cleavage and polyadenylation at the 3′ endof the RNA; Cbp80p, involved in capping the 5′ end of the RNA; andNp13p, a homolog of mammalian hnRNP A1, involved in export of mRNA fromthe nucleus (Shen, E. C. et al. (1998) Genes Dev. 12:679-691). HnRNPshave been shown to be important targets of the autoimmune response inrheumatic diseases (Biamonti, supra).

Many snRNP and hnRNP proteins are characterized by an RNA recognitionmotif (RRM). (Reviewed in Birney, E. et al. (1993) Nucleic Acids Res.21:5803-5816.) The RRM is about 80 amino acids in length and forms fourβ-strands and two α-helices arranged in an α/β sandwich. The RRMcontains a core RNP-1 octapeptide motif along with surrounding conservedsequences. In addition to snRNP proteins, examples of RNA-bindingproteins which contain the above motifs include heteronuclearribonucleoproteins which stabilize nascent RNA and factors whichregulate alternative splicing. Alternative splicing factors includedevelopmentally regulated proteins, specific examples of which have beenidentified in lower eukaryotes such as Drosophila melanogaster andCaenorhabditis elegans. These proteins play key roles in developmentalprocesses such as pattern formation and sex determination, respectively.(See, for example, Hodgkin, J. et al. (1994) Development 120:3681-3689.)

The 3′ ends of most eukaryote mRNAs are also posttranscriptionallymodified by polyadenylation. Polyadenylation proceeds through twoenzymatically distinct steps: (i) the endonucleolytic cleavage ofnascent mRNAs at cis-acting polyadenylation signals in the3′-untranslated (non-coding) region and (ii) the addition of a poly(A)tract to the 5′ mRNA fragment. The presence of cis-acting RNA sequencesis necessary for both steps. These sequences include 5′-AAUAAA-3′located 10-30 nucleotides upstream of the cleavage site and a lesswell-conserved GU- or U-rich sequence element located 10-30 nucleotidesdownstream of the cleavage site. Cleavage stimulation factor (CstF),cleavage factor I (CF I), and cleavage factor II (CF II) are involved inthe cleavage reaction while cleavage and polyadenylation specificityfactor (CPSF) and poly(A) polymerase (PAP) are necessary for bothcleavage and polyadenylation. An additional enzyme, poly(A)-bindingprotein II (PAB II), promotes poly(A) tract elongation (Rüegsegger, U.et al. (1996) J. Biol. Chem. 271:6107-6113; and references within).

Translation

Correct translation of the genetic code depends upon each amino acidforming a linkage with the appropriate transfer RNA (tRNA). Theaminoacyl-tRNA synthetases (aaRSs) are essential proteins found in allliving organisms. The aaRSs are responsible for the activation andcorrect attachment of an amino acid with its cognate tRNA, as the firststep in protein biosynthesis. Prokaryotic organisms have at least twentydifferent types of aaRSs, one for each different amino acid, whileeukaryotes usually have two aaRSs, a cytosolic form and a mitochondrialform, for each different amino acid. The 20 aaRS enzymes can be dividedinto two structural classes. Class I enzymes add amino acids to the 2′hydroxyl at the 3′ end of tRNAs while Class II enzymes add amino acidsto the 3′ hydroxyl at the 3′ end of tRNAs. Each class is characterizedby a distinctive topology of the catalytic domain. Class I enzymescontain a catalytic domain based on the nucleotide-binding Rossman‘fold’. In particular, a consensus tetrapeptide motif is highlyconserved (Prosite Document PDOC00161, Aminoacyl-transfer RNAsynthetases class-I signature). Class I enzymes are specific forarginine, cysteine, glutarnic acid, glutamine, isoleucine, leucine,methionine, tyrosine, tryptophan, and valine. Class II enzymes contain acentral catalytic domain, which consists of a seven-strandedantiparallel β-sheet domain, as well as N- and C-terminal regulatorydomains. Class II enzymes are separated into two groups based on theheterodimeric or homodimeric structure of the enzyme; the latter groupis further subdivided by the structure of the N- and C-terminalregulatory domains (Hartlein, M. and Cusack, S. (1995) J. Mol. Evol.40:519-530). Class II enzymes are specific for alanine, asparagine,aspartic acid, glycine, histidine, lysine, phenylalanine, proline,serine, and threonine.

Certain aaRSs also have editing functions. IleRS, for example, canmisactivate valine to form Val-tRNA^(Ile), but this product is clearedby a hydrolytic activity that destroys the mischarged product. Thisediting activity is located within a second catalytic site found in theconnective polypeptide 1 region (CP1), a long insertion sequence withinthe Rossman fold domain of Class I enzymes (Schimmel, P. et al. (1998)FASEB J. 12:1599-1609). AaRSs also play a role in tRNA processing. Ithas been shown that mature tRNAs are charged with their respective aminoacids in the nucleus before export to the cytoplasm, and charging mayserve as a quality control mechanism to insure the tRNAs are functional(Martinis, S. A. et al. (1999) EMBO J. 18:4591-4596).

Under optimal conditions, polypeptide synthesis proceeds at a rate ofapproximately 40 amino acid residues per second. The rate ofmisincorporation during translation in on the order of 10⁻⁴ and isprimarily the result of aminoacyl-t-RNAs being charged with theincorrect amino acid. Incorrectly charged tRNA are toxic to cells asthey result in the incorporation of incorrect amino acid residues intoan elongating polypeptide. The rate of translation is presumed to be acompromise between the optimal rate of elongation and the need fortranslational fidelity. Mathematical calculations predict that 10⁻⁴ isindeed the maximum acceptable error rate for protein synthesis in abiological system (reviewed in Stryer, supra; and Watson, J. et al.(1987) The Benjamin/Cummings Publishing Co., Inc. Menlo Park, Calif.). Aparticularly error prone aminoacyl-tRNA charging event is the chargingof tRNA^(Gln), with Gln. A mechanism exits for the correction of thismischarging event which likely has its origins in evolution. Gin wasamong the last of the 20 naturally occurring amino acids used inpolypeptide synthesis to appear in nature. Gram positive eubacteria,cyanobacteria, Archeae, and eukaryotic organelles possess a noncanonicalpathway for the synthesis of Gln-tRNA^(Gin) based on the transformationof Glu-tRNA^(Gln) (synthesized by Glu-tRNA synthetase, GluRS) using theenzyme Glu-tRNA^(Gin) amidotransferase (Glu-AdT). The reactions involvedin the transamidation pathway are as follows (Curnow, A. W. et al.(1997) Nucleic Acids Symposium 36:2-4):

A similar enzyme, Asp-tRNA^(Asn) amidotransferase, exists in Archaea,which transforms Asp-tRNA^(Asn) to Asn-tRNA^(Asn). Formylase, the enzymethat transforms Met-tRNA^(fMet) to fMet-tRNA^(fMet) in eubacteria, islikely to be a related enzyme. A hydrolytic activity has also beenidentified that destroys mischarged Val-tRNA^(Ile) (Schimmel, P. et al.(1998) FASEB J. 12:1599-1609). One likely scenario for the evolution ofGlu-AdT in primitive life forms is the absence of a specificglutaminyl-tRNA synthetase (GlnRS), requiring an alternative pathway forthe synthesis of Gln-tRNA^(Gln). In fact, deletion of the Glu-AdT operonin Gram positive bacteria is lethal (Curnow, A. W. et al. (1997) Proc.Natl. Acad. Sci. USA 94:11819-11826). The existence of GluRS activity inother organisms has been inferred by the high degree of conservation intranslation machinery in nature; however, GluRS has not been identifiedin all organisms, including Homo sapiens. Such an enzyme would beresponsible for ensuring translational fidelity and reducing thesynthesis of defective polypeptides.

In addition to their function in protein synthesis, specific aminoacyltRNA synthetases also play roles in cellular fidelity, RNA splicing, RNAtrafficking, apoptosis, and transcriptional and translationalregulation. For example, human tyrosyl-tRNA synthetase can beproteolytically cleaved into two fragments with distinct cytokineactivities. The carboxy-terminal domain exhibits monocyte and leukocytechemotaxis activity as well as stimulating production ofmyeloperoxidase, tumor necrosis factor-α, and tissue factor. TheN-terminal domain binds to the interleukin-8 type A receptor andfunctions as an interleukin-8-like cytokine. Human tyrosyl-tRNAsynthetase is secreted from apoptotic tumor cells and may accelerateapoptosis (Wakasugi, K., and Schimmel, P. (1999) Science 284:147-151).Mitochondrial Neurospora crassa TyrRS and S. cerevisiae LeuRS areessential factors for certain group I intron splicing activities, andhuman mitochondrial LeuRS can substitute for the yeast LeuRS in a yeastnull strain. Certain bacterial aaRSs are involved in regulating theirown transcription or translation (Martinis, supra). Several aaRSs areable to synthesize diadenosine oligophosphates, a class of signallingmolecules with roles in cell proliferation, differentiation, andapoptosis (Kisselev, L. L et al. (1998) FEBS Lett. 427:157-163;Vartanian, A. et al. (1999) FEBS Lett. 456:175-180).

Autoantibodies against aminoacyl-tRNAs are generated by patients withautoimmune diseases such as rheumatic arthritis, dermatomyositis andpolymyositis, and correlate strongly with complicating interstitial lungdisease (ILD) (Freist, W. et al. (1999) Biol. Chem. 380:623-646; Freist,W. et al. (1996) Biol. Chem. Hoppe Seyler 377:343-356). These antibodiesappear to be generated in response to viral infection, and coxsackievirus has been used to induce experimental viral myositis in animals.

Comparison of aaRS structures between humans and pathogens has beenuseful in the design of novel antibiotics (Schimmel, supra). Geneticallyengineered aaRSs have been utilized to allow site-specific incorporationof unnatural amino acids into proteins in vivo (Liu, D. R. et al. (1997)Proc. Natl. Acad. Sci. USA 94:10092-10097).

tRNA Modifications

The modified ribonucleoside, pseudouridine (ψ), is present ubiquitouslyin the anticodon regions of transfer RNAs (tRNAs), large and smallribosomal RNAs (rRNAs), and small nuclear RNAs (snRNAs). y is the mostcommon of the modified nucleosides (i.e., other than G, A, U, and C)present in tRNAs. Only a few yeast tRNAs that are not involved inprotein synthesis do not contain ψ (Cortese, R. et al. (1974) J. Biol.Chem. 249:1103-1108). The enzyme responsible for the conversion ofuridine to ψ, pseudouridine synthase (pseudouridylate synthase), wasfirst isolated from Salmonella typhimurium (Arena, F. et al. (1978)Nucleic Acids Res. 5:4523-4536). The enzyme has since been isolated froma number of mammals, including steer and mice (Green, C. J. et al.(1982) J. Biol. Chem. 257:3045-52; and Chen, J. and Patton, J. R. (1999)RNA 5:409-419). tRNA pseudouridine synthases have been the mostextensively studied members of the family. They require a thiol donor(e.g., cysteine) and a monovalent cation (e.g., ammonia or potassium)for optimal activity. Additional cofactors or high energy molecules(e.g., ATP or GTP) are not required (Green, supra). Other eukaryoticpseudouridine synthases have been identified that appear to be specificfor rRNA (reviewed in Smith, C. M. and Steitz, J. A. (1997) Cell89:669-672) and a dual-specificity enzyme has been identified that usesboth tRNA and rRNA substrates (Wrzesinski, J. et al. (1995) RNA 1:437-448). The absence of ψ in the anticodon loop of tRNAs results inreduced growth in both bacteria (Singer, C. E. et al. (1972) Nature NewBiol. 238:72-74) and yeast (Lecointe, F. (1998) J. Biol. Chem.273:1316-1323), although the genetic defect is not lethal.

Another ribonucleoside modification that occurs primarily in eukaryoticcells is the conversion of guanosine to N²,N²-dimethylguanosine (m² ₂G)at position 26 or 10 at the base of the D-stem of cytosolic andmitochondrial tRNAs. This posttranscriptional modification is believedto stabilize tRNA structure by preventing the formation of alternativetRNA secondary and tertiary structures. Yeast tRNA^(Asp) is unusual inthat it does not contain this modification. The modification does notoccur in eubacteria, presumably because the structure of tRNAs in thesecells and organelles is sequence constrained and does not requireposttranscriptional modification to prevent the formation of alternativestructures (Steinberg, S. and Cedergren, R. (1995) RNA 1:886-891, andreferences within). The enzyme responsible for the conversion ofguanosine to m² ₂G is a 63 kDa S-adenosylmethionine (SAM)-dependent tRNAN²,N²-dimethyl-guanosine methyltransferase (also referred to as the TRM1gene product and herein referred to as TRM) (Edqvist, J. (1995)Biochimie 77:54-61). The enzyme localizes to both the nucleus and themitochondria (Li, J-M. et al. (1989) J. Cell Biol. 109:1411-1419). Basedon studies with TRM from Xenopus laevis, there appears to be arequirement for base pairing at positions C11-G24 and G10-C25immediately preceding the G26 to be modified, with other structuralfeatures of the tRNA also being required for the proper presentation ofthe G26 substrate (Edqvist. J. et al. (1992) Nucleic Acids Res.20:6575-6581). Studies in yeast suggest that cells carrying a weak ochretRNA suppressor (sup3-i) are unable to suppress translation terminationin the absence of TRM activity, suggesting a role for TRM in modifyingthe frequency of suppression in eukaryotic cells (Niederberger, C. etal. (1999) FEBS Lett. 464:67-70), in addition to the more generalfunction of ensuring the proper three-dimensional structures for tRNA.

Translation Initiation

Initiation of translation can be divided into three stages. The firststage brings an initiator transfer RNA (Met-tRNA_(f)) together with the40S ribosomal subunit to form the 43S preinitiation complex. The secondstage binds the 43S preinitiation complex to the mRNA, followed bymigration of the complex to the correct AUG initiation codon. The thirdstage brings the 60S ribosomal subunit to the 40S subunit to generate an80S ribosome at the inititation codon. Regulation of translationprimarily involves the first and second stage in the initiation process(V. M. Pain (1996) Eur. J. Biochem. 236:747-771).

Several initiation factors, many of which contain multiple subunits, areinvolved in bringing an initiator tRNA and the 40S ribosomal subunittogether. eIF2, a guanine nucleotide binding protein, recruits theinitiator tRNA to the 40S ribosomal subunit. Only when eIF2 is bound toGTP does it associate with the initiator tRNA. eIF2B, a guaninenucleotide exchange protein, is responsible for converting eIF2 from theGDP-bound inactive form to the GTP-bound active form. Two other factors,eIFIA and eIF3 bind and stabilize the 40S subunit by interacting withthe 18S ribosomal RNA and specific ribosomal structural proteins. eIF3is also involved in association of the 40S ribosomal subunit with mRNA.The Met-tRNA_(f), eIF1A, eIF3, and 40S ribosomal subunit together makeup the 43S preinitiation complex (Pain, supra).

eIF2 plays a central role in the maintenance of a rate-limiting step inmRNA translation. In this step, eIF2 binds GTP and Met-tRNAi andtransfers Met-tRNAi to the 40S ribosomal subunit. At the end of theinitiation process, GTP bound to eIF2 is hydrolyzed to GDP and theeIF2.GDP complex is released from the ribosome. The exchange of GDPbound to eIF2 for GTP is a prerequisite to binding Met-tRNAi and ismediated by a second initiation factor, eIF2B, a guaninenucleotide-exchange factor. Phosphorylation of eIF2 on its alpha-subunitconverts eIF2 from a substrate of eIF2B into a competitive inhibitor.Thus, phosphorylation of eIF2 alpha effectively prevents formation ofthe e[F2.GTP.Met-tRNAi complex and inhibits global protein synthesis.Phosphorylation of eIF2 alpha occurs under a variety of conditionsincluding viral infection, apoptosis, nutrient deprivation,heme-deprivation, and certain stresses. The 5′-untranslated region ofhepatitis C virus (HCV) functions as an internal ribosome entry site(IRES) to initiate translation of HCV proteins. eIF2Bgamma and eIF2gammaare cellular factors involved in HCV IRES-mediated translation (Kimball,S. R. (1999) Int. J. Biochem. Cell Biol. 31:25-29; Webb, B. L. andProud, C. G. (1997) Int. J. Biochem. Cell Biol. 29:1127-1131; Kruger M.et al. (2000) Proc. Natl. Acad. Sci. U S A 97:8566-8571).

Additional factors are required for binding of the 43S preinitiationcomplex to an mRNA molecule, and the process is regulated at severallevels. eIF4F is a complex consisting of three proteins: eIF4E, eIF4A,and eIF4G. eIF4E recognizes and binds to the mRNA 5′-terminal m⁷GTP cap,eIF4A is a bidirectional RNA-dependent helicase, and eIF4G is ascaffolding polypeptide. eIF4G has three binding domains. The N-terminalthird of eIF4G interacts with eIF4E, the central third interacts witheIF4A, and the C-terminal third interacts with eIF3 bound to the 43Spreinitiation complex. Thus, eIF4G acts as a bridge between the 40Sribosomal subunit and the mRNA (M. W. Hentze (1997) Science275:500-501).

The ability of eIF4F to initiate binding of the 43S preinitiationcomplex is regulated by structural features of the mRNA. The mRNAmolecule has an untranslated region (UTR) between the 5′ cap and the AUGstart codon. In some mRNAs this region forms secondary structures thatimpede binding of the 43S preinitiation complex. The helicase activityof eIF4A is thought to function in removing this secondary structure tofacilitate binding of the 43S preinitiation complex (Pain, supra).

Translation Elongation

Elongation is the process whereby additional amino acids are joined tothe initiator methionine to form the complete polypeptide chain. Theelongation factors EF1 α, EF2 β γ, and EF2 are involved in elongatingthe polypeptide chain following initiation. EF1 α is a GTP-bindingprotein. In EF1 α's GTP-bound form, it brings an aminoacyl-tRNA to theribosome's A site. The amino acid attached to the newly arrivedaminoacyl-tRNA forms a peptide bond with the initiator methionine. TheGTP on EF1 α is hydrolyzed to GDP, and EF1 α-GDP dissociates from theribosome. EF1 β γ binds EF1 α-GDP and induces the dissociation of GDPfrom EF1 α allowing EF1 α to bind GTP and a new cycle to begin.

As subsequent aminoacyl-tRNAs are brought to the ribosome, EF-G, anotherGTP-binding protein, catalyzes the translocation of tRNAs from the Asite to the P site and finally to the E site of the ribosome. Thisallows the ribosome and the mRNA to remain attached during translation.

The MCM domain is found in DNA-dependent ATPases required for theinitiation of eukaryotic DNA replication. In eukaryotes there is afamily of six proteins that contain this domain, MCM2 to MCM7 (Hu, B. etal. (1993) Nucleic Acids Res. 21:5289-5293).

Translation Termination

The release factor eRF carries out termination of translation. eRFrecognizes stop codons in the mRNA, leading to the release of thepolypeptide chain from the ribosome.

The apical ectodermal ridge (AER) is an essential structure forvertebrate limb development. Wnt3a is expressed during the induction ofchick AER. Misexpression of Wnt3a induces ectopic expression ofAER-specific genes in the limb ectoderm. The genes beta-catenin and Lef1mimic the effect of Wnt3a. Blocking the intrinsic Lef1 activity disruptsAER formation. Hence, Wnt3a functions in AER formation through thebeta-catenin/LEF1 pathway. In contrast, neither beta-catenin nor Lef1affects the Wnt7a-regulated dorsoventral polarity of the limb. Thus, tworelated Wnt genes elicit distinct responses in the same tissues by usingdifferent intracellular pathways (Kengaku, M. et al.(1998) Science280:1274-1277).

Treacher Collins Syndrome (TCS) is the most common of the humanmandibulofacial dysostosis disorders. It shows autosomal dominantinheritance and occurs in 1 of 50,000 live births, with approximately60% arising from new mutations. TCS symptoms show wide variability. Thedisease is deduced to be a result of interference in the development ofthe first and second branchial arches. The TCS gene, TCOF1, is localizedto chromosome 5q31-33.3. There are ten identified mutations in TCOF1consisting of nonsense mutations, insertions, deletions, or splicingmutations that apparently lead to premature termination of translation.Moreover, all are unique to each human family. TCOF1 encodes a lowcomplexity protein of 1,411 amino acids, with repeated motifs thatmirror the organization of its exons. These motifs are shared withnucleolar trafficking proteins in other species and are highlyphosphorylated by casein kinase. The full-length TCOF1 protein sequencealso contains nuclear and nucleolar localization signals and severalpolymorphisms. This data suggests that TCS results from defects in anucleolar trafficking protein that is critically required during humancraniofacial development (Wise, C. A. et al. (1997) Proc. Natl. Acad.Sci. U.S.A. 94:3110-3115).

Breast Cancer

There are more than 180,000 new cases of breast cancer diagnosed eachyear, and the mortality rate for breast cancer approaches 10% of alldeaths in females between the ages of 45-54 (Gish, K. (1999) AWISMagazine 28:7-10). However the survival rate based on early diagnosis oflocalized breast cancer is extremely high (97%), compared with theadvanced stage of the disease in which the tumor has spread beyond thebreast (22%). Current procedures for clinical breast examination arelacking in sensitivity and specificity, and efforts are underway todevelop comprehensive gene expression profiles for breast cancer thatmay be used in conjunction with conventional screening methods toimprove diagnosis and prognosis of this disease (Perou, C. M. et al.(2000) Nature 406:747-752).

Mutations in two genes, BRCA1 and BRCA2, are known to greatly predisposea woman to breast cancer and may be passed on from parents to children(Gish, supra). However, this type of hereditary breast cancer accountsfor only about 5% to 9% of breast cancers, while the vast majority ofbreast cancer is due to non-inherited mutations that occur in breastepithelial cells.

The relationship between expression of epidermal growth factor (EGF) andits receptor, EGFR, to human mammary carcinoma has been particularlywell studied. (See Khazaie, K. et al. (1993) Cancer and Metastasis Rev.12:255-274, and references cited therein for a review of this area.)Overexpression of EGFR, particularly coupled with down-regulation of theestrogen receptor, is a marker of poor prognosis in breast cancerpatients. In addition, EGFR expression in breast tumor metastases isfrequently elevated relative to the primary tumor, suggesting that EGFRis involved in tumor progression and metastasis. This is supported byaccumulating evidence that EGF has effects on cell functions related tometastatic potential, such as cell motility, chemotaxis, secretion anddifferentiation. Changes in expression of other members of the erbBreceptor family, of which EGFR is one, have also been implicated inbreast cancer. The abundance of erbB receptors, such as HER-2/neu,HER-3, and HER-4, and their ligands in breast cancer points to theirfunctional importance in the pathogenesis of the disease, and maytherefore provide targets for therapy of the disease (Bacus, S. S. etal. (1994) Am. J. Clin. Pathol. 102:S13-S24). Other known markers ofbreast cancer include a human secreted frizzled protein mRNA that isdownregulated in breast tumors; the matrix G1a protein which isoverexpressed is human breast carcinoma cells; Drg1 or RTP, a gene whoseexpression is diminished in colon, breast, and prostate tumors; maspin,a tumor suppressor gene downregulated in invasive breast carcinomas; andCaN19, a member of the S100 protein family, all of which are downregulated in mammary carcinoma cells relative to normal mammaryepithelial cells (Zhou, Z. et al. (1998) Int. J. Cancer 78:95-99; Chen,L. et al. (1990) Oncogene 5:1391-1395; Ulrix, W. et al (1999) FEBS Lett455:23-26; Sager, R. et al. (1996) Curr. Top. Microbiol. Immunol.213:51-64; and Lee, S. W. et al. (1992) Proc. Natl. Acad. Sci. USA89:2504-2508).

Cell lines derived from human mammary epithelial cells at various stagesof breast cancer provide a useful model to study the process ofmalignant transformation and tumor progression as it has been shown thatthese cell lines retain many of the properties of their parental tumorsfor lengthy culture periods (Wistuba, I. I. et al. (1998) Clin. CancerRes. 4:2931-2938). Such a model is particularly useful for comparingphenotypic and molecular characteristics of human mammary epithelialcells at various stages of malignant transformation.

Preadipocyte Cells

The most important function of adipose tissue is its ability to storeand release fat during periods of feeding and fasting. White adiposetissue is the major energy reserve in periods of excess energy use, andits primary purpose is mobilization during energy deprivation.Understanding how the various molecules regulate adiposity and energybalance in physiological and pathophysiological situations may lead tothe development of novel therapeutics for human obesity. Adipose tissueis also one of the important target tissues for insulin. Adipogenesisand insulin resistance in type II diabetes are linked and presentintriguing relations. Most patients with type II diabetes are obese andobesity in turn causes insulin resistance.

The majority of research in adipocyte biology to date has been doneusing transformed mouse preadipocyte cell lines. The culture condition,which stimulates mouse preadipocyte differentiation is different fromthat for inducing human primary preadipocyte differentiation. Inaddition, primary cells are diploid and may therefore reflect the invivo context better than aneuploid cell lines. Understanding the geneexpression profile during adipogenesis in human will lead tounderstanding the fundamental mechanism of adiposity regulation.Furthermore, through comparing the gene expression profiles ofadipogenesis between donor with normal weight and donor with obesity,identification of crucial genes, potential drug targets for obesity andtype II diabetes, will be possible.

Peroxisome Proliferator-Activated Receptor Gamma Agonist

Thiazolidinediones (IZDs) act as agonists for theperoxisome-proliferator-activated receptor gamma (PPARγ), a member ofthe nuclear hormone receptor superfamily. TZDs reduce hyperglycemia,hyperinsulinemia, and hypertension, in part by promoting glucosemetabolism and inhibiting gluconeogenesis. Roles for PPARγ and itsagonists have been demonstrated in a wide range of pathologicalconditions including diabetes, obesity, hypertension, atherosclerosis,polycystic ovarian syndrome, and cancers such as breast, prostate,liposarcoma, and colon cancer.

The mechanism by which IZDs and other PPARγ agonists enhance insulinsensitivity is not fully understood, but may involve the ability ofPPARγ to promote adipogenesis. When ectopically expressed in culturedpreadipocytes, PPARγ is a potent inducer of adipocyte differentiation.IZDs, in combination with insulin and other factors, can also enhancedifferentiation of human preadipocytes in culture (Adams et al. (1997)J. Clin. Invest. 100:3149-3153). The relative potency of different mIDsin promoting adipogenesis in vitro is proportional to both their insulinsensitizing effects in vivo, and their ability to bind and activatePPARγ in vitro. Interestingly, adipocytes derived from omental adiposedepots are refractory to the effects of TZDs. It has therefore beensuggested that the insulin sensitizing effects of lZDs may result fromtheir ability to promote adipogenesis in subcutaneous adipose depots(Adams et al., ibid). Further, dominant negative mutations in the PPARγgene have been identified in two non-obese subjects with severe insulinresistance, hypertension, and overt non-insulin dependent diabetesmellitus (NIDDM) (Barroso et al. (1998) Nature 402:880-883).

NIDDM is the most common form of diabetes meffitus, a chronic metabolicdisease that affects 143 million people worldwide. NIDDM ischaracterized by abnormal glucose and lipid metabolism that result froma combination of peripheral insulin resistance and defective insulinsecretion. NIDDM has a complex, progressive etiology and a high degreeof heritability. Numerous complications of diabetes including heartdisease, stroke, renal failure, retinopathy, and peripheral neuropathycontribute to the high rate of morbidity and mortality.

At the molecular level, PPARγ functions as a ligand activatedtranscription factor. In the presence of ligand, PPARγ forms aheterodimer with the retinoid X receptor (RXR) which then activatestranscription of target genes containing one or more copies of a PPARγresponse element (PPRE). Many genes important in lipid storage andmetabolism contain PPREs and have been identified as PPARγ targets,including PEPCK, aP2, LPL, ACS, and FAT-P (Auwerx, J. (1999)Diabetologia 42:1033-1049). Multiple ligands for PPARγ have beenidentified. These include a variety of fatty acid metabolites; syntheticdrugs belonging to the TZD class, such as Pioglitazone and Rosiglitazone(BRLA9653); and certain non-glitazone tyrosine analogs such as G1262570and GW1929. The prostaglandin derivative 15-dPGJ2 is a potent endogenousligand for PPARγ.

Expression of PPARγ is very high in adipose but barely detectable inskeletal muscle, the primary site for insulin stimulated glucosedisposal in the body. PPARγ is also moderately expressed in largeintestine, kidney, liver, vascular smooth muscle, hematopoietic cells,and macrophages. The high expression of PPARγ in adipose suggests thatthe insulin sensitizing effects of TZDs may result from alterations inthe expression of one or more PPARγ regulated genes in adipose tissue.Identification of PPARγ target genes will contribute to better drugdesign and the development of novel therapeutic strategies for diabetes,obesity, and other conditions.

Systematic attempts to identify PPARγ target genes have been made inseveral rodent models of obesity and diabetes (Suzuki et al. (2000) Jpn.J. Pharmacol. 84:113-123; Way et al. (2001) Endocrinology142:1269-1277). However, a serious drawback of the rodent geneexpression studies is that significant differences exist between humanand rodent models of adipogenesis, diabetes, and obesity (Taylor (1999)Cell 97:9-12; Gregoire et al. (1998) Physiol. Reviews 78:783-809).Therefore, an unbiased approach to identifying TZD regulated genes inprimary cultures of human tissues is necessary to fully elucidate themolecular basis for diseases associated with PPARγ activity.

Lung Cancer

Lung cancer is the leading cause of cancer death for men and the secondleading cause of cancer death for women in the U.S. The vast majority oflung cancer cases are attributed to smoking tobacco, and increased useof tobacco products in third world countries is projected to lead to anepidemic of lung cancer in these countries. Exposure of the bronchialepithelium to tobacco smoke appears to result in changes in tissuemorphology, which are thought to be precursors of cancer. Lung cancersare divided into four histopathologically distinct groups. Three groups(squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) areclassified as non-small cell lung cancers (NSCLCs). The fourth group ofcancers is referred to as small cell lung cancer (SCLC). Collectively,NSCLCs account for ˜70% of cases while SCLCs account for ˜18% of cases.The molecular and cellular biology underlying the development andprogression of lung cancer are incompletely understood.

Deletions on chromosome 3 are common in this disease and are thought toindicate the presence of a tumor suppressor gene in this region.Activating mutations in K-ras are commonly found in lung cancer and arethe basis of one of the mouse models for the disease.

Colorectal Cancer

Colorectal cancer is the second leading cause of cancer deaths in theUnited States, and is thought to be a disease of aging since 90% of thetotal cases occur in individuals over the age of 55. A widely acceptedhypothesis is that several mutations must accumulate over time in anindividual who develops the disease. To understand the nature of genealterations in colorectal cancer, a number of studies have focused onthe inherited syndromes. The first, Familial Adenomatous Polyposis(FAP), is caused by mutations in the Adenomatous Polyposis Coli gene(APC), resulting in truncated or inactive forms of the protein. Thistumor suppressor gene has been mapped to chromosome 5q. The second knowninherited syndrome is hereditary nonpolyposis colorectal cancer (HNPCC),which is caused by mutations in mismatch repair genes. Althoughhereditary colon cancer syndromes occur in a small percentage of thepopulation, and most colorectal cancers are considered sporadic,knowledge from studies of the hereditary syndromes can be appliedbroadly. For instance, somatic mutations in APC occur in at least 80% ofsporadic colon tumors. APC mutations are thought to be the initiatingevent in disease progression. Other mutations occur subsequently.Approximately 50% of colorectal cancers contain activating mutations inras, while 85% contain inactivating mutations in p53. Changes in all ofthese genes lead to gene expression changes in colon cancer.

Ovarian Cancer

Ovarian cancer is the leading cause of death from a gynecologic cancer.The majority of ovarian can-cers are derived from epithelial cells, and70% of patients with epithelial ovarian cancers present with late-stagedisease. Identification of early-stage markers for ovarian cancer wouldsignificantly increase the survival rate. Some of the molecular eventsimplicated in ovarian cancer include mutation of p53 and niicrosatelliteinstability.

Additional Diseases and Related Factors

Tangier disease (TD) is a genetic disorder characterized by near absenceof circulating HDL and the accumulation of cholesterol esters in manytissues, including tonsils, lymph nodes, liver, spleen, thymus, andintestine. Low levels of HDL represent a clear predictor of prematurecoronary artery disease and homozygous TD correlates with a four- tosix-fold increase in cardiovascular disease compared to controls. Themajor cardioprotective activity of HDL is ascribed to its role inreverse cholesterol transport, the flux of cholesterol from peripheralcells such as tissue macrophages, through plasma lipoproteins to theliver. The HDL protein, apolipoprotein AI plays a major role in thisprocess, interacting with the cell surface to remove excess cholesteroland phospholipids. This pathway is severely impaired in TD. The defectlies in a specific gene, the ABC1 transporter. This gene is a member ofthe family of ATP-binding cassette transporters, which utilize ATPhydrolysis to transport a variety of substrates across membranes.

The effects upon liver metabolism and hormone clearance mechanisms areimportant to understand the pharmacodynamics of a drug. The human C3Acell line is a clonal derivative of HepG2/C3 (hepatoma cell line,isolated from a 15-year-old male with liver tumor), which was selectedfor strong contact inhibition of growth. The use of a clonal populationenhances the reproducibility of the cells. C3A cells have manycharacteristics of primary human hepatocytes in culture: i) expressionof insulin receptor and insulin-like growth factor II receptor; ii)secretion of a high ratio of serum albumin compared with a-fetoproteiniii) conversion of ammonia to urea and glutamine;, iv) metabolism ofaromatic amino acids; and v) proliferation in glucose-free andinsulin-free medium. The C3A cell line is now well established as an invitro model of the mature human liver (Mickelson et al. (1995)Hepatology 22:866-875; Nagendra et al. (1997) Am J Physiol272:G408-G416).

Dexamethasone (DEX) is a synthetic glucocorticoid used as ananti-inflammatory or immuno-suppressive agent. Due to its greaterability to reach the central nervous system, DEX is usually thetreatment of choice to control cerebral edema. Glucocorticoids arenaturally occurring hormones that prevent or suppress inflammation andimmune responses when administered at pharmacological doses. At themolecular level, unbound glucocorticoids readily cross cell membranesand bind with high affinity to specific cytoplasmic receptors.Subsequent to binding, transcription and protein synthesis are affected.The result can include inhibition of leukocyte infiltration at the siteof inflammation, interference in the function of mediators ofinflammatory response, and suppression of humoral immune responses. Theanti-inflammatory actions of corticosteroids are thought to involvephospholipase A 2 inhibitory proteins, collectively called lipocortins.Lipocortins, in turn, control the biosynthesis of potent mediators ofinflammation such as prostaglandins and leukotrienes by inliubiting therelease of the precursor molecule arachidonic acid.

Human aortic endothelial cells (HMVECdNeos) are primary cells derivedfrom the endothelium of the microvasculature of human skin. HMVECdNeoshave been used as an experimental model for investigating in vitro therole of the endothelium in human vascular biology. Activation of thevascular endothelium is considered a central event in a wide range ofboth physiological and pathophysiological processes, such as vasculartone regulation, coagulation and thrombosis, atherosclerosis, andinflammation.

Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that playsa central role in mediation of the inflammatory response throughactivation of multiple signal transduction pathways. TNF-α is producedby activated lymphocytes, macrophages, and other white blood cells, andis known to activate endothelial cells. Monitoring the endothelial cellresponse to TNF-α at the level of mRNA expression can provideinformation necessary for better understanding of both TNF-α signalingand endothelial cell biology.

Dendritic cells (DCs), as antigen presenting cells, play a crucial rolein the initiation of the immune response. DCs can be derived in vitroeither from CD34+ bone marrow precursors (IDCs) or from peripheral bloodmonocytic cells (mDCs). In vivo, DCs reside in two distinctcompartments: the peripheral tissues such as lung, skin, kidney, heart,and intestine; and in secondary lymphoid organs such as lymph node,spleen, and Peyer's patches. In the periphery, DCs are efficient antigenprocessing cells but are limited in their capacity to activate naive Tcells. Upon activation (injury, inflammation, infection), DCs entertheir final stage of maturation during which they downregulate thecapacity to process new antigens, migrate out of the periphery into thesecondary lymphoid organs, and acquire an extremely potent capacity toactivate naive T cells. Factors such as cross linking the CD40 surfacemolecules or the presence of TNF-α can induce this final stage ofmaturation.

CD40 is a type I integral membrane glycoprotein belonging to theTNF-receptor family. It is expressed on all mature B lymphocytes,dendritic cells, and some epithelial cells. Antibodies specific for CD40molecules can induce proliferation of B cells when presented with EL-4or antibodies specific for CD20 molecules. Also, stimulation of B cellswith anti-CD40 antibodies and IL-4 can induce the switch ofimmunoglobulin production to the IgE isotype.

Characterization of region-specific gene expression in the human brainprovides a context and background for molecular neurobiology research ingeneral. Information from RNA expression in these tissues may supplyinsight into the genetic basis of brain structure and function, whichmay in turn become useful in drug target discovery.

Array technology can provide a simple way to explore the expression of asingle polymorphic gene or the expression profile of a large number ofrelated or unrelated genes. When the expression of a single gene isexamined, arrays are employed to detect the expression of a specificgene or its variants. When an expression profile is examined, arraysprovide a platform for examining which genes are tissue specific,carrying out housekeeping functions, parts of a signaling cascade, orspecifically related to a particular genetic predisposition, condition,disease, or disorder. The potential application of gene expressionprofiling is particularly relevant to improving diagnosis, prognosis,and treatment of disease. For example, both the levels and sequencesexpressed in tissues from subjects with diabetes may be compared withthe levels and sequences expressed in normal tissue.

Expression Profiling

Microarrays are analytical tools used in bioanalysis. A microarray has aplurality of molecules spatially distributed over, and stably associatedwith, the surface of a solid support. Microarrays of polypeptides,polynucleotides, and/or antibodies have been developed and find use in avariety of applications, such as gene sequencing, monitoring geneexpression, gene mapping, bacterial identification, drug discovery, andcombinatorial chemistry.

One area in particular in which microarrays find use is in geneexpression analysis. Array technology can provide a simple way toexplore the expression of a single polymorphic gene or the expressionprofile of a large number of related or unrelated genes. When theexpression of a single gene is examined, arrays are employed to detectthe expression of a specific gene or its variants. When an expressionprofile is examined, arrays provide a platform for identifying genesthat are tissue specific, are affected by a substance being tested in atoxicology assay, are part of a signaling cascade, carry outhousekeeping functions, or are specifically related to a particulargenetic predisposition, condition, disease, or disorder.

There is a need in the art for new compositions, including nucleic acidsand proteins, for the diagnosis, prevention, and treatment of cellproliferative, neurological, developmental, and autoimmune/inflammatorydisorders, and infections.

SUMMARY OF THE INVENTION

Various embodiments of the invention provide purified polypeptides,nucleic acid-associated proteins, referred to collectively as ‘NAAP’ andindividually as ‘NAAP-1,’ ‘NAAP-2,’ ‘NAAP-3,’ ‘NAAP-4,’ ‘NAAP-5,’‘NAAP-6,’ ‘NAAP-7,’ ‘NAAP-8,’ ‘NAAP-9,’ ‘NAAP10,’ ‘NAAP-11,’ ‘NAAP-12,’‘NAAP-13,’ ‘NAAP-14,’ ‘NAAP-15,’ ‘NAAP-16,’ ‘NAAP-17,’ ‘NAAP-18,’‘NAAP-19,’ ‘NAAP-20,’ ‘NAAP-21,’ ‘NAAP-22,’ ‘NAAP-23,’ ‘NAAP-24,’‘NAAP-25,’ ‘NAAP-26,’ ‘NAAP-27,’ ‘NAAP-28,’ ‘NAAP-29,’ ‘NAAP-30,’‘NAAP-31,’ ‘NAAP-32,’ NAAP-33,’ ‘NAAP-34,’ ‘NAAP-35,’ ‘NAAP-36,’‘NAAP-37,’ ‘NAAP-38,’ ‘NAAP-39,’ ‘NAAP-40,’ ‘NAAP-41,’ ‘NAAP-42,’‘NAAP-43,’ ‘NAAP-44,’ ‘NAAP-45,’ ‘NAAP-46,’ ‘NAAP-47,’ ‘NAAP-48,’‘NAAP-49,’ ‘NAAP-50,’ ‘NAAP-51,’ ‘NAAP-52,’ ‘NAAP-53,’ ‘NAAP-54,’‘NAAP-55,’ ‘NAAP-56,’ ‘NAAP-57,’ ‘and ‘NAAP-58’ and methods for usingthese proteins and their encoding polynucleotides for the detection,diagnosis, and treatment of diseases and medical conditions. Embodimentsalso provide methods for utilizing the purified nucleic acid-associatedproteins and/or their encoding polynucleotides for facilitating the drugdiscovery process, including determination of efficacy, dosage,toxicity, and pharmacology. Related embodiments provide methods forutilizing the purified nucleic acid-associated proteins and/or theirencoding polynucleotides for investigating the pathogenesis of diseasesand medical conditions.

An embodiment provides an isolated polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-58, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-58. Another embodiment provides anisolated polypeptide comprising an amino acid sequence of SEQ IDNO:1-58.

Still another embodiment provides an isolated polynucleotide encoding apolypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-58, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ IDNO:1-58, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-58, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-58. Inanother embodiment, the polynucleotide encodes a polypeptide selectedfrom the group consisting of SEQ ID NO:1-58. In an alternativeembodiment, the polynucleotide is selected from the group consisting ofSEQ ID NO:59-116.

Still another embodiment provides a recombinant polynucleotidecomprising a promoter sequence operably linked to a polynucleotideencoding a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58. Another embodiment provides a celltransformed with the recombinant polynucleotide. Yet another embodimentprovides a transgenic organism comprising the recombinantpolynucleotide.

Another embodiment provides a method for producing a polypeptideselected from the group consisting of a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ IDNO:1-58, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical or at least about 90% identical to anamino acid sequence selected from the group consisting of SEQ IDNO:1-58, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-58, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-58. Themethod comprises a) culturing a cell under conditions suitable forexpression of the polypeptide, wherein said cell is transformed with arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide encoding the polypeptide, and b) recoveringthe polypeptide so expressed.

Yet another embodiment provides an isolated antibody which specificallybinds to a polypeptide selected from the group consisting of a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, b) a polypeptide comprising a naturallyoccurring amino acid sequence at least 90% identical or at least about90% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, c) a biologically active fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58.

Still yet another embodiment provides an isolated polynucleotideselected from the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:59-116, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:59-116, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). In otherembodiments, the polynucleotide can comprise at least about 20, 30, 40,60, 80, or 100 contiguous nucleotides.

Yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:59-116, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:59-116, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) hybridizing the sample with a probe comprising at least 20contiguous nucleotides comprising a sequence complementary to saidtarget polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex. In a related embodiment, themethod can include detecting the amount of the hybridization complex. Instill other embodiments, the probe can comprise at least about 20, 30,40, 60, 80, or 100 contiguous nucleotides.

Still yet another embodiment provides a method for detecting a targetpolynucleotide in a sample, said target polynucleotide being selectedfrom the group consisting of a) a polynucleotide comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:59-116, b) a polynucleotide comprising a naturally occurringpolynucleotide sequence at least 90% identical or at least about 90%identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:59-116, c) a polynucleotide complementary to thepolynucleotide of a), d) a polynucleotide complementary to thepolynucleotide of b), and e) an RNA equivalent of a)-d). The methodcomprises a) amplifying said target polynucleotide or fragment thereofusing polymerase chain reaction amplification, and b) detecting thepresence or absence of said amplified target polynucleotide or fragmentthereof. In a related embodiment, the method can include detecting theamount of the amplified target polynucleotide or fragment thereof.

Another embodiment provides a composition comprising an effective amountof a polypeptide selected from the group consisting of a) a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1-58, b) a polypeptide comprising a naturally occurring aminoacid sequence at least 90% identical or at least about 90% identical toan amino acid sequence selected from the group consisting of SEQ IDNO:1-58, c) a biologically active fragment of a polypeptide having anamino acid sequence selected from the group consisting of SEQ IDNO:1-58, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence selected from the group consisting of SEQ ID NO:1-58, anda pharmaceutically acceptable excipient. In one embodiment, thecomposition can comprise an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58. Other embodiments provide a method oftreating a disease or condition associated with decreased or abnormalexpression of functional NAAP, comprising administering to a patient inneed of such treatment the composition.

Yet another embodiment provides a method for screening a compound foreffectiveness as an agonist of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-58, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-58. The method comprises a) exposinga sample comprising the polypeptide to a compound, and b) detectingagonist activity in the sample. Another embodiment provides acomposition comprising an agonist compound identified by the method anda pharmaceutically acceptable excipient. Yet another embodiment providesa method of treating a disease or condition associated with decreasedexpression of functional NAAP, comprising administering to a patient inneed of such treatment the composition.

Still yet another embodiment provides a method for screening a compoundfor effectiveness as an antagonist of a polypeptide selected from thegroup consisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-58, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-58. The method comprises a) exposinga sample comprising the polypeptide to a compound, and b) detectingantagonist activity in the sample. Another embodiment provides acomposition comprising an antagonist compound identified by the methodand a pharmaceutically acceptable excipient. Yet another embodimentprovides a method of treating a disease or condition associated withoverexpression of functional NAAP, comprising administering to a patientin need of such treatment the composition.

Another embodiment provides a method of screening for a compound thatspecifically binds to a polypeptide selected from the group consistingof a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-58, b) a polypeptide comprising anaturally occurring amino acid sequence at least 90% identical or atleast about 90% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NO:1-58, c) a biologically active fragment ofa polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58, and d) an immunogenic fragment of apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58. The method comprises a) combining thepolypeptide with at least one test compound under suitable conditions,and b) detecting binding of the polypeptide to the test compound,thereby identifying a compound that specifically binds to thepolypeptide.

Yet another embodiment provides a method of screening for a compoundthat modulates the activity of a polypeptide selected from the groupconsisting of a) a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, b) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical or at least about 90% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO:1-58, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-58, and d) an immunogenicfragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO:1-58. The method comprises a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound,and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide.

Still yet another embodiment provides a method for screening a compoundfor effectiveness in altering expression of a target polynucleotide,wherein said target polynucleotide comprises a polynucleotide sequenceselected from the group consisting of SEQ ID NO:59-116, the methodcomprising a) exposing a sample comprising the target polynucleotide toa compound, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.

Another embodiment provides a method for assessing toxicity of a testcompound, said method comprising a) treating a biological samplecontaining nucleic acids with the test compound; b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide selected from thegroup consisting of i) a polynucleotide comprising a polynucleotidesequence selected from the group consisting of SEQ ID NO:59-116, ii) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical or at least about 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:59-116, iii) a polynucleotide having a sequence complementary to i),iv) a polynucleotide complementary to the polynucleotide of ii), and v)an RNA equivalent of i)-iv). Hybridization occurs under conditionswhereby a specific hybridization complex is formed between said probeand a target polynucleotide in the biological sample, said targetpolynucleotide selected from the group consisting of i) a polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:59-116, ii) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical or at leastabout 90% identical to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO:59-116, iii) a polynucleotide complementary tothe polynucleotide of i), iv) a polynucleotide complementary to thepolynucleotide of ii), and v) an RNA equivalent of i)-iv).Alternatively, the target polynucleotide can comprise a fragment of apolynucleotide selected from the group consisting of i)-v) above; c)quantifying the amount of hybridization complex; and d) comparing theamount of hybridization complex in the treated biological sample withthe amount of hybridization complex in an untreated biological sample,wherein a difference in the amount of hybridization complex in thetreated biological sample is indicative of toxicity of the testcompound.

BRIEF DESCRIPTION OF THE TABLES

Table 1 summarizes the nomenclature for full length polynucleotide andpolypeptide embodiments of the invention.

Table 2 shows the GenBank identification number and annotation of thenearest GenBank homolog, and the PROTEOME database identificationnumbers and annotations of PROTEOME database homologs, for polypeptideembodiments of the invention. The probability scores for the matchesbetween each polypeptide and its homolog(s) are also shown.

Table 3 shows structural features of polypeptide embodiments, includingpredicted motifs and domains, along with the methods, algorithms, andsearchable databases used for analysis of the polypeptides.

Table 4 lists the cDNA and/or genomic DNA fragments which were used toassemble polynucleotide embodiments, along with selected fragments ofthe polynucleotides.

Table 5 shows representative cDNA libraries for polynucleotideembodiments.

Table 6 provides an appendix which describes the tissues and vectorsused for construction of the cDNA libraries shown in Table 5.

Table 7 shows the tools, programs, and algorithms used to analyzepolynucleotides and polypeptides, along with applicable descriptions,references, and threshold parameters.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleic acids, and methods are described,it is understood that embodiments of the invention are not limited tothe particular machines, instruments, materials, and methods described,as these may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a host cell” includes aplurality of such host cells, and a reference to “an antibody” is areference to one or more antibodies and equivalents thereof known tothose skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with variousembodiments of the invention. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

Definitions

“NAAP” refers to the amino acid sequences of substantially purified NAAPobtained from any species, particularly a mammalian species, includingbovine, ovine, porcine, murine, equine,, and human, and from any source,whether natural, synthetic, semi-synthetic, or recombinant.

The term “agonist” refers to a molecule which intensifies or mimics thebiological activity of NAAP. Agonists may include proteins, nucleicacids, carbohydrates, small molecules, or any other compound orcomposition which modulates the activity of NAAP either by directlyinteracting with NAAP or by acting on components of the biologicalpathway in which NAAP participates.

An “allelic variant” is an alternative form of the, gene encoding NAAP.Allelic variants may result from at least one mutation in the nucleicacid sequence and may result in altered mRNAs or in polypeptides whosestructure or function may or may not be altered. A gene may have none,one, or many allelic variants of its naturally occurring form. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Altered” nucleic acid sequences encoding NAAP include those sequenceswith deletions, insertions, or substitutions of different nucleotides,resulting in a polypeptide the same as NAAP or a polypeptide with atleast one functional characteristic of NAAP. Included within thisdefinition are polymnorphisms which may or may not be readily detectableusing a particular oligonucleotide probe of the polynucleotide encodingNAAP, and improper or unexpected hybridization to allelic variants, witha locus other than the normal chromosomal locus for the polynucleotideencoding NAAP. The encoded protein may also be “altered,” and maycontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentNAAP. Deliberate amino acid substitutions may be made on the basis ofone or more similarities in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of NAAP isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, and positively charged amino acids mayinclude lysine and arginine. Amino acids with uncharged polar sidechains having similar hydrophilicity values may include: asparagine andglutamine; and serine and threonine. Amino acids with uncharged sidechains having similar hydrophilicity values may include: leucine,isoleucine, and valiue; glycine and alanine; and phenylalanine andtyrosine.

The terms “amino acid” and “amino acid sequence” can refer to anoligopeptide, a peptide, a polypeptide, or a protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. Where “amino acid sequence” is recited to refer to a sequenceof a naturally occurring protein molecule, “amino acid sequence” andlike terms are not meant to limit the amino acid sequence to thecomplete-native amino acid sequence associated with the recited proteinmolecule.

“Amplification” relates to the production of additional copies of anucleic acid. Amplification may be carried out using polymerase chainreaction (PCR) technologies or other nucleic acid amplificationtechnologies well known in the art.

The term “antagonist” refers to a molecule which inhibits or attenuatesthe biological activity of NAAP. Antagonists may include proteins suchas antibodies, anticalins, nucleic acids, carbohydrates, smallmolecules, or any other compound or composition which modulates theactivity of NAAP either by directly interacting with NAAP or by actingon components of the biological pathway in which NAAP participates.

The term “antibody” refers to intact immunoglobulin molecules as well asto fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding an epitopic determinant. Antibodies that bind NAAPpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to inmmunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant” refers to that region of a molecule(i.e., an epitope) that makes contact with a particular antibody. When aprotein or a fragment of a protein is used to immunize a host animal,numerous regions of the protein may induce the production of antibodieswhich bind specifically to antigenic determinants (particular regions orthree-dimensional structures on the protein). An antigenic determinantmay compete with the intact antigen (i.e., the immunogen used to elicitthe immune response) for binding to an antibody.

The term “aptamer” refers to a nucleic acid or oligonucleotide moleculethat binds to a specific molecular target. Aptamers are derived from anin vitro evolutionary process (e.g., SELEX (Systematic Evolution ofLigands by Exponential Enrichment), described in U.S. Pat. No.5,270,163), which selects for target-specific aptamer sequences fromlarge combinatorial libraries. Aptamer compositions may bedouble-stranded or single-stranded, and may includedeoxyribonucleotides, ribonucleotides, nucleotide derivatives, or othernucleotide-like molecules. The nucleotide components of an aptamer mayhave modified sugar groups (e.g., the 2′-OH group of a ribonucleotidemay be replaced by 2′-F or 2′-NH₂), which may improve a desiredproperty, e.g., resistance to nucleases or longer lifetime in blood.Aptamers may be conjugated to other molecules, e.g., a high molecularweight carrier to slow clearance of the aptamer from the circulatorysystem. Aptamers may be specifically cross-linked to their cognateligands, e.g., by photo-activation of a cross-linker (Brody, E. N. andL. Gold (2000) J. Biotechnol. 74:5-13).

The term “intramer” refers to an aptamer which is expressed in vivo. Forexample, a vaccinia virus-based RNA expression system has been used toexpress specific RNA aptamers at high levels in the cytoplasm ofleukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA96:3606-3610).

The term “spiegelmer” refers to an aptamer which includes L-DNA, L-RNA,or other left-handed nucleotide derivatives or nucleotide-likemolecules. Aptamers containig left-handed nucleotides are resistant todegradation by naturally occurring enzymes, which normally act onsubstrates containing right-handed nucleotides.

The term “antisense” refers to any composition capable of base-pairingwith the “sense” (coding) strand of a polynucleotide having a specificnucleic acid sequence. Antisense compositions may include DNA; RNA;peptide nucleic acid (PNA); oligonucleotides having modified backbonelinkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; oligonucleotides having modified sugar groups suchas 2′-methoxyethyl sugars or 2′-methoxyethoxy sugars; oroligonucleotides having modified bases such as 5-methyl cytosine,2′-deoxyuracil, or 7-deaza-2′-deoxyguanosine. Antisense molecules may beproduced by any method including chemical synthesis or transcription.Once introduced into a cell, the complementary antisense moleculebase-pairs with a naturally occurring nucleic acid sequence produced bythe cell to form duplexes which block either transcription ortranslation. The designation “negative” or “minus” can refer to theantisense strand, and the designation “positive” or “plus” can refer tothe sense strand of a reference DNA molecule.

The term “biologically active” refers to a protein having structural,regulatory, or biochemical functions of a naturally occurring molecule.Likewise, “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic NAAP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

“Complementary” describes the relationship between two single-strandednucleic acid sequences that anneal by base-pairing. For example,5′-AGT-3′ pairs with its complement, 3′-TCA-5′.

A “composition comprising a given polynucleotide” and a “compositioncomprising a given polypeptide” can refer to any composition containingthe given polynucleotide or polypeptide. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotides encoding NAAP or fragments of NAAP may be employed ashybridization probes. The probes may be stored in freeze-dried form andmay be associated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate;SDS), and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

“Consensus sequence” refers to a nucleic acid sequence which has beensubjected to repeated DNA sequence analysis to resolve uncalled bases,extended using the XL-PCR kit (Applied Biosystems, Foster City Calif.)in the 5′ and/or the 3′ direction, and resequenced, or which has beenassembled from one or more overlapping cDNA, EST, or genormic DNAfragments using a computer program for fragment assembly, such as theGELVIEW fragment assembly system (Accelrys, Burlington Mass.) or Phrap(University of Washington, Seattle Wash.). Some sequences have been bothextended and assembled to produce the consensus sequence.

“Conservative amino acid substitutions” are those substitutions that arepredicted to least interfere with the properties of the originalprotein, i.e., the structure and especially the function of the proteinis conserved and not significantly changed by such substitutions. Thetable below shows amino acids which may be substituted for an originalamino acid in a protein and which are regarded as conservative aminoacid substitutions. Original Residue Conservative Substitution Ala Gly,Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln Asn,Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gln, Glu Ile Leu, ValLeu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met, Leu, Trp, TyrSer Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe, Trp Val Ile, Leu,Thr

Conservative amino acid substitutions generally maintain (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a beta sheet or alpha helical conformation, (b) thecharge or hydrophobicity of the molecule at the site of thesubstitution, and/or (c) the bulk of the side chain.

A “deletion” refers to a change in the amino acid or nucleotide sequencethat results in the absence of one or more amino acid residues ornucleotides.

The term “derivative” refers to a chemically modified polynucleotide orpolypeptide. Chemical modifications of a polynucleotide can include, forexample, replacement of hydrogen by an alkyl, acyl, hydroxyl, or aminogroup. A derivative polynucleotide encodes a polypeptide which retainsat least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

A “detectable label” refers to a reporter molecule or enzyme that iscapable of generating a measurable signal and is covalently ornoncovalently joined to a polynucleotide or polypeptide.

“Differential expression” refers to increased or upregulated; ordecreased, downregulated, or absent gene or protein expression,determined by comparing at least two different samples. Such comparisonsmay be carried out between, for example, a treated and an untreatedsample, or a diseased and a normal sample.

“Exon shuffling” refers to the recombination of different coding regions(exons). Since an exon may represent a structural or functional domainof the encoded protein, new proteins may be assembled through the novelreassortment of stable substructures, thus allowing acceleration of theevolution of new protein functions.

A “fragment” is a unique portion of NAAP or a polynucleotide encodingNAAP which can be identical in sequence to, but shorter in length than,the parent sequence. A fragment may comprise up to the entire length ofthe defined sequence, minus one nucleotide/amino acid residue. Forexample, a fragment may comprise from about 5 to about 1000 contiguousnucleotides or amino acid residues. A fragment used as a probe, primer,antigen, therapeutic molecule, or for other purposes, may be at least 5,10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500contiguous nucleotides or amino acid residues in length. Fragments maybe preferentially selected from certain regions of a molecule. Forexample, a polypeptide fragment may comprise a certain length ofcontiguous amino acids selected from the first 250 or 500 amino acids(or first 25% or 50%) of a polypeptide as shown in a certain definedsequence. Clearly these lengths are exemplary, and any length that issupported by the specification, including the Sequence Listing, tables,and figures, may be encompassed by the present embodiments.

A fragment of SEQ ID NO:59-116 can comprise a region of uniquepolynucleotide sequence that specifically identifies SEQ ID NO:59-116,for example, as distinct from any other sequence in the genome fromwhich the fragment was obtained. A fragment of SEQ ID NO:59-116 can beemployed in one or more embodiments of methods of the invention, forexample, in hybridization and amplification technologies and inanalogous methods that distinguish SEQ ID NO:59-116 from relatedpolynucleotides. The precise length of a fragment of SEQ ID NO:59-116and the region of SEQ ID NO:59-116 to which the fragment corresponds areroutinely determinable by one of ordinary skill in the art based on theintended purpose for the fragment.

A fragment of SEQ ID NO:1-58 is encoded by a fragment of SEQ IDNO:59-116. A fragment of SEQ ID NO:1-58 can comprise a region of uniqueamino acid sequence that specifically identifies SEQ ID NO:1-58. Forexample, a fragment of SEQ ID NO:1-58 can be used as an immunogenicpeptide for the development of antibodies that specifically recognizeSEQ ID NO:1-58. The precise length of a fragment of SEQ ID NO:1-58 andthe region of SEQ ID NO:1-58 to which the fragment corresponds can bedetermined based on the intended purpose for the fragment using one ormore analytical methods described herein or otherwise known in the art.

A “full length” polynucleotide is one containing at least a translationinitiation codon (e.g., methionine) followed by an open reading frameand a translation termination codon. A “full length” polynucleotidesequence encodes a “full length” polypeptide sequence.

“Homology” refers to sequence similarity or, alternatively, sequenceidentity, between two or more polynucleotide sequences or two or morepolypeptide sequences.

The terms “percent identity” and “% identity,” as applied topolynucleotide sequences, refer to the percentage of identical residuematches between at least two polynucleotide sequences aligned using astandardized algorithm. Such an algorithm may insert, in a standardizedand reproducible way, gaps in the sequences being compared in order tooptimize alignment between two sequences, and therefore achieve a moremeaningful comparison of the two sequences.

Percent identity between polynucleotide sequences may be determinedusing one or more computer algorithms or programs known in the art ordescribed herein. For example, percent identity can be determined usingthe default parameters of the CLUSTAL V algorithm as incorporated intothe MEGALIGN version 3.12e sequence alignment program. This program ispart of the LASERGENE software package, a suite of molecular biologicalanalysis programs (DNASTAR, Madison Wis.). CLUSTAL V is described inHiggins, D. G. and P. M. Sharp (1989; CABIOS 5:151-153) and in Higgins,D. G. et al. (1992; CABIOS 8:189-191). For pairwise alignments ofpolynucleotide sequences, the default parameters are set as follows:Ktuple=2, gap penalty=5, window=4, and “diagonals saved”=4. The“weighted” residue weight table is selected as the default.

Alternatively, a suite of commonly used and freely available sequencecomparison algorithms which can be used is provided by the NationalCenter for Biotechnology Information (NCBI) Basic Local Alignment SearchTool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410),which is available from several sources, including the NCBI, Bethesda,Md., and on the Internet at http://www.ncbi.nlm.nih.gov/BLAST/. TheBLAST software suite includes various sequence analysis programsincluding “blastn,” that is used to align a known polynucleotidesequence with other polynucleotide sequences from a variety ofdatabases. Also available is a tool called “BLAST 2 Sequences” that isused for direct pairwise comparison of two nucleotide sequences. “BLAST2 Sequences” can be accessed and used interactively athttp://www.ncbi.nlm.nih.gov/gorf/b12.html. The “BLAST 2 Sequences” toolcan be used for both blastn and blastp (discussed below). BLAST programsare commonly used with gap and other parameters set to default settings.For example, to compare two nucleotide sequences, one may use blastnwith the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set atdefault parameters. Such default parameters maybe, for example:

-   -   Matrix: BLOSUM62    -   Reward for match: 1    -   Penalty for mismatch: −2    -   Open Gap: 5 and Extension Gap: 2 penalties    -   Gap×drop-off: 50    -   Expect: 10    -   Word Size: 11    -   Filter: on

Percent identity may be measured over the length of an entire definedsequence, for example, as defined by a particular SEQ ID number, or maybe measured over a shorter length, for example, over the length of afragment taken from a larger, defined sequence, for instance, a fragmentof at least 20, at least 30, at least 40, at least 50, at least 70, atleast 100, or at least 200 contiguous nucleotides. Such lengths areexemplary only, and it is understood that any fragment length supportedby the sequences shown herein, in the tables, figures, or SequenceListing, may be used to describe a length over which percentage identitymay be measured.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences due to the degeneracyof the genetic code. It is understood that changes in a nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences that all encode substantially the same protein.

The phrases “percent identity” and “% identity,” as applied topolypeptide sequences, refer to the percentage of identical residuematches between at least two polypeptide sequences aligned using astandardized algorithm. Methods of polypeptide sequence alignment arewell-known. Some alignment methods take into account conservative aminoacid substitutions. Such conservative substitutions, explained in moredetail above, generally preserve the charge and hydrophobicity at thesite of substitution, thus preserving the structure (and thereforefunction) of the polypeptide. The phrases “percent similarity” and “%similarity,” as applied to polypeptide sequences, refer to thepercentage of residue matches, including identical residue matches andconservative substitutions, between at least two polypeptide sequencesaligned using a standardized algorithm. In contrast, conservativesubstitutions are not included in the calculation of percent identitybetween polypeptide sequences.

Percent identity between polypeptide sequences may be determined usingthe default parameters of the CLUSTAL V algoritun as incorporated intothe MEGALIGN version 3.12e sequence alignment program (described andreferenced above). For pairwise alignments of polypeptide sequencesusing CLUSTAL V, the default parameters are set as follows: Ktuple=1,gap penalty=3, window=5, and “diagonals saved”=5. The PAM250 matrix isselected as the default residue weight table.

Alternatively the NCBI BLAST software suite may be used. For example,for a pairwise comparison of two polypeptide sequences, one may use the“BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp setat default parameters. Such default parameters may be, for example:

-   -   Matrix: BLOSUM62    -   Open Gap: 11 and Extension Gap: 1 penalties    -   Gap×drop-off: 50    -   Expect: 10    -   Word Size: 3    -   Filter: on

Percent identity may be measured over the length of an entire definedpolypeptide sequence, for example, as defined by a particular SEQ IDnumber, or may be measured over a shorter length, for example, over thelength of a fragment taken from a larger, defined polypeptide sequence,for instance, a fragment of at least 15, at least 20, at least 30, atleast 40, at least 50, at least 70 or at least 150 contiguous residues.Such lengths are exemplary only, and it is understood that any fragmentlength supported by the sequences shown herein, in the tables, figuresor Sequence Listing, may be used to describe a length over whichpercentage identity may be measured.

“Human artificial chromosomes” (HACs) are linear microchromosomes whichmay contain DNA sequences of about 6 kb to 10 Mb in size and whichcontain all of the elements required for chromosome replication,segregation and maintenance.

The term “humanized antibody” refers to an antibody molecule in whichthe amino acid sequence in the non-antigen binding regions has beenaltered so that the antibody more closely resembles a human antibody,and still retains its original binding ability.

“Hybridization” refers to the process by which a polynucleotide strandanneals with a complementary strand through base pairing under definedhybridization conditions. Specific hybridization is an indication thattwo nucleic acid sequences share a high degree of complementarity.Specific hybridization complexes form under permissive annealingconditions and remain hybridized after the “washing” step(s). Thewashing step(s) is particularly important in determining the stringencyof the hybridization process, with more stringent conditions allowingless non-specific binding, i.e., binding between pairs of nucleic acidstrands that are not perfectly matched. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and may be consistent among hybridizationexperiments, whereas wash conditions may be varied among experiments toachieve the desired stringency, and therefore hybridization specificity.Permissive annealing conditions occur, for example, at 68° C. in thepresence of about 6×SSC, about 1% (w/v) SDS, and about 100 μg/mlsheared, denatured salmon sperm DNA.

Generally, stringency of hybridization is expressed, in part, withreference to the temperature under which the wash step is carried out.Such wash temperatures are typically selected to be about 5° C. to 20°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An equation forcalculating T_(m) and conditions for nucleic acid hybridization are wellknown and can be found in Sambrook, J. and D. W. Russell (2001;Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold SpringHarbor Press, Cold Spring Harbor N.Y., ch. 9).

High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68° C. in the presenceof about 0.2×SSC and about 0.1% SDS, for 1 hour. Alternatively,temperatures of about 65° C., 60° C., 55° C., or 42° C. may be used. SSCconcentration may be varied from about 0.1 to 2×SSC, with SDS beingpresent at about 0.1%. Typically, blocking reagents are used to blocknon-specific hybridization. Such blocking reagents include, forinstance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml.Organic solvent, such as formamide at a concentration of about 35-50%v/v, may also be used under particular circumstances, such as forRNA:DNA hybridizations. Useful variations on these wash conditions willbe readily apparent to those of ordinary skill in the art.Hybridization, particularly under high stringency conditions, may besuggestive of evolutionary similarity between the nucleotides. Suchsimilarity is strongly indicative of a similar role for the nucleotidesand their encoded polypeptides.

The term “hybridization complex” refers to a complex formed between twonucleic acids by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(e.g., Cot or Rot analysis) or formed between one nucleic acid presentin solution and another nucleic acid immobilized on a solid support(e.g., paper, membranes, filters, chips, pins or glass slides, or anyother appropriate substrate to which cells or their nucleic acids havebeen fixed).

The words “insertion” and “addition” refer to changes in an amino acidor polynucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

An “immunogenic fragment” is a polypeptide or oligopeptide fragment ofNAAP which is capable of eliciting an immune response when introducedinto a living organism, for example, a mammal. The term “immunogenicfragment” also includes any polypeptide or oligopeptide fragment of NAAPwhich is useful in any of the antibody production methods disclosedherein or known in the art.

The term “microarray” refers to an arrangement of a plurality ofpolynucleotides, polypeptides, antibodies, or other chemical compoundson a substrate.

The terms “element” and “array element” refer to a polynucleotide,polypeptide, antibody, or other chemical compound having a unique anddefined position on a microarray.

The term “modulate” refers to a change in the activity of NAAP. Forexample, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or inmmunological properties of NAAP.

The phrases “nucleic acid” and “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material.

“Operably linked” refers to the situation in which a first nucleic acidsequence is placed in a functional relationship with a second nucleicacid sequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be in close proximityor contiguous and, where necessary to join two protein coding regions,in the same reading frame.

“Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

“Post-translational modification” of an NAAP may involve lipidation,glycosylation, phosphorylation, acetylation, racemization, proteolyticcleavage, and other modifications known in the art. These processes mayoccur synthetically or biochemically. Biochemical modifications willvary by cell type depending on the enzymatic milieu of NAAP.

“Probe” refers to nucleic acids encoding NAAP, their complements, orfragments thereof, which are used to detect identical, allelic orrelated nucleic acids. Probes are isolated oligonucleotides orpolynucleotides attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, ligands, chemiluminescentagents, and enzymes. “Primers” are short nucleic acids, usually DNAoligonucleotides, which may be annealed to a target polynucleotide bycomplementary base-pairing. The primer may then be extended along thetarget DNA strand by a DNA polymerase enzyme. Primer pairs can be usedfor amplification (and identification) of a nucleic acid, e.g., by thepolymerase chain reaction (PCR).

Probes and primers as used in the present invention typically compriseat least 15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of thedisclosed nucleic acid sequences. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the specification, including the tables, figures, andSequence Listing, may be used.

Methods for preparing and using probes and primers are described in, forexample, Sambrook, J. and D. W. Russell (2001; Molecular Cloning: ALaboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, ColdSpring Harbor N.Y.), Ausubel, F. M. et al. (1999; Short Protocols inMolecular Biology, 4th ed., John Wiley & Sons, New York N.Y.), andInnis, M. et al. (1990; PCR Protocols, A Guide to Methods andApplications, Academic Press, San Diego Calif.). PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers are selected using software known inthe art for such purpose. For example, OLIGO 4.06 software is useful forthe selection of PCR primer pairs of up to 100 nucleotides each, and forthe analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope. The Primer3 primerselection program (available to the public from the WhiteheadInstitute/MIT Center for Genome Research, Cambridge Mass.) allows theuser to input a “mispriming library,” in which sequences to avoid asprimer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed above.

A “recombinant nucleic acid” is a nucleic acid that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo or more otherwise separated segments of sequence. This artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, e.g., by genetic engineering techniques such as those describedin Sambrook and Russell (supra). The term recombinant includes nucleicacids that have been altered solely by addition, substitution, ordeletion of a portion of the nucleic acid. Frequently, a recombinantnucleic acid may include a nucleic acid sequence operably linked to apromoter sequence. Such a recombinant nucleic acid may be part of avector that is used, for example, to transform a cell.

Alternatively, such recombinant nucleic acids may be part of a viralvector, e.g., based on a vaccinia virus, that could be use to vaccinatea mammal wherein the recombinant nucleic acid is expressed, inducing aprotective immunological response in the mammal.

A “regulatory element” refers to a nucleic acid sequence usually derivedfrom untranslated regions of a gene and includes enhancers, promoters,introns, and 5′ and 3′ untranslated regions (UTRs). Regulatory elementsinteract with host or viral proteins which control transcription,translation, or RNA stability.

“Reporter molecules” are chemical or biochemical moieties used forlabeling a nucleic acid, amino acid, or antibody. Reporter moleculesinclude radionuclides; enzymes; fluorescent, chemiluminescent, orchromogenic agents; substrates; cofactors; inhibitors; magneticparticles; and other moieties known in the art.

An “RNA equivalent,” in reference to a DNA molecule, is composed of thesame linear sequence of nucleotides as the reference DNA molecule withthe exception that all occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The term “sample” is used in its broadest sense. A sample suspected ofcontaining NAAP, nucleic acids encoding NAAP, or fragments thereof maycomprise a bodily fluid; an extract from a cell, chromosome, organelle,or membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA, insolution or bound to a substrate; a tissue; a tissue print; etc.

The terms “specific binding” and “specifically binding” refer to thatinteraction between a protein or peptide and an agonist, an antibody, anantagonist, a small molecule, or any natural or synthetic bindingcomposition. The interaction is dependent upon the presence of aparticular structure of the protein, e.g., the antigenic determinant orepitope, recognized by the binding molecule. For example, if an antibodyis specific for epitope “A,” the presence of a polypeptide comprisingthe epitope A, or the presence of free unlabeled A, in a reactioncontaining free labeled A and the antibody will reduce the amount oflabeled A that binds to the antibody.

The term “substantially purified” refers to nucleic acid or amino acidsequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably atleast about 75% free, and most preferably at least about 90% free fromother components with which they are naturally associated.

A “substitution” refers to the replacement of one or more amino acidresidues or nucleotides by different amino acid residues or nucleotides,respectively.

“Substrate” refers to any suitable rigid or semi-rigid support includingmembranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

A “transcript image” or “expression profile” refers to the collectivepattern of gene expression by a particular cell type or tissue undergiven conditions at a given time.

“Transformation” describes a process by which exogenous DNA isintroduced into a recipient cell. Transformation may occur under naturalor artificial conditions according to various methods well known in theart, and may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod for transformation is selected based on the type of host cellbeing transformed and may include, but is not limited to, bacteriophageor viral infection, electroporation, heat shock, lipofection, andparticle bombardment. The term “transformed cells” includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “transgenic organism,” as used herein, is any organism, including butnot limited to animals and plants, in which one or more of the cells ofthe organism contains heterologous nucleic acid introduced by way ofhuman intervention, such as by transgenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. In another embodiment, the nucleicacid can be introduced by infection with a recombinant viral vector,such as a lentiviral vector (Lois, C. et al. (2002) Science295:868-872). The term genetic manipulation does not include classicalcross-breeding, or in vitro fertilization, but rather is directed to theintroduction of a recombinant DNA molecule. The transgenic organismscontemplated in accordance with the present invention include bacteria,cyanobacteria, fungi, plants and animals. The isolated DNA of thepresent invention can be introduced into the host by methods known inthe art, for example infection, transfection, transformation ortransconjugation. Techniques for transferring the DNA of the presentinvention into such organisms are widely known and provided inreferences such as Sambrook and Russell (supra).

A “variant” of a particular nucleic acid sequence is defined as anucleic acid sequence having at least 40% sequence identity to theparticular nucleic acid sequence over a certain length of one of thenucleic acid sequences using blastn with the “BLAST 2 Sequences” toolVersion 2.0.9 (May 7, 1999) set at default parameters. Such a pair ofnucleic acids may show, for example, at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% or greater sequence identityover a certain defined length. A variant may be described as, forexample, an “allelic” (as defined above), “splice,” “species,” or“polymorphic” variant. A splice variant may have significant identity toa reference molecule, but will generally have a greater or lesser numberof polynucleotides due to alternate splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or lack domains that are present in the referencemolecule. Species variants are polynucleotides that vary from onespecies to another. The resulting polypeptides will generally havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass “single nucleotide polymorphisms” (SNPs) in which thepolynucleotide sequence varies by one nucleotide base. The presence ofSNPs may be indicative of, for example, a certain population, a diseasestate, or a propensity for a disease state.

A “variant” of a particular polypeptide sequence is defined as apolypeptide sequence having at least 40% sequence identity or sequencesirnilarity to the particular polypeptide sequence over a certain lengthof one of the polypeptide sequences using blastp with the “BLAST 2Sequences” tool Version 2.0.9 (May 7, 1999) set at default parameters.Such a pair of polypeptides may show, for example, at least 50%, atleast 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% or greatersequence identity or sequence similarity over a certain defined lengthof one of the polypeptides.

The Invention

Various embodiments of the invention include new human nucleicacid-associated proteins (NAAP), the polynucleotides encoding NAAP, andthe use of these compositions for the diagnosis, treatment, orprevention of cell proliferative, neurological, developmental, andautoimmune/inflammatory disorders, and infections.

Table 1 summarizes the nomenclature. for the full length polynucleotideand polypeptide embodiments of the invention. Each polynucleotide andits corresponding polypeptide are correlated to a single Incyte projectidentification number (Incyte Project ID). Each polypeptide sequence isdenoted by both a polypeptide sequence identification number(Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number(Incyte Polypeptide ID) as shown. Each polynucleotide sequence isdenoted by both a polynucleotide sequence identification number(Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensussequence number (Incyte Polynucleotide ID) as shown. Column 6 shows theIncyte ID numbers of physical, full length clones corresponding to thepolypeptide and polynucleotide sequences of the invention. The fulllength clones encode polypeptides which have at least 95% sequenceidentity to the polypeptide sequences shown in column 3.

Table 2 shows sequences with homology to polypeptide embodiments of theinvention as identified by BLAST analysis against the GenBank protein(genpept) database and the PROTEOME database. Columns 1 and 2 show thepolypeptide sequence identification number (Polypeptide SEQ ID NO:) andthe corresponding Incyte polypeptide sequence number (Incyte PolypeptideID) for polypeptides of the invention. Column 3 shows the GenBankidentification number (GenBank ID NO:) of the nearest GenBank homologand the PROTEOME database identification numbers (PROTEOME ID NO:) ofthe nearest PROTEOME database homologs. Column 4 shows the probabilityscores for the matches between each polypeptide and its homolog(s).Column 5 shows the annotation of the GenBank and PROTEOME databasehomolog(s) along with relevant citations where applicable, all of whichare expressly incorporated by reference herein.

Table 3 shows various structural features of the polypeptides of theinvention. Columns 1 and 2 show the polypeptide sequence identificationnumber (SEQ ID NO:) and the corresponding Incyte polypeptide sequencenumber (Incyte Polypeptide ID) for each polypeptide of the invention.Column 3 shows the number of amino acid residues in each polypeptide.Column 4 shows potential phosphorylation sites, and column 5 showspotential glycosylation sites, as determined by the MOTIFS program ofthe GCG sequence analysis software package (Accelrys, Burlington Mass.).Column 6 shows amino acid residues comprising signature sequences,domains, and motifs. Column 7 shows analytical methods for proteinstructure/function analysis and in some cases, searchable databases towhich the analytical methods were applied.

Together, Tables 2 and 3 summarize the properties of polypeptides of theinvention, and these properties establish that the claimed polypeptidesare nucleic acid-associated proteins. For example, SEQ ID NO:2 is 57%identical, from residue T192 to residue T586, to human DNA bindingprotein (GenBank ID g1020145) as determined by the Basic Local AlignmentSearch Tool (BLAST). (See Table 2.) The BLAST probability score is1.1e-149, which indicates the probability of obtaining the observedpolypeptide sequence alignment by chance. SEQ ID NO:2 is localized tothe nucleus, binds DNA, and is a zinc finger protein containing a KRABdomain, as determined by BLAST analysis using the PROTEOME database. SEQID NO:2 also contains a KRAB box domain and 14 zinc finger, C2H2 type,domains as determined by searching for statistically significant matchesin the hidden Markov model (HMM)-based PFAM database of conservedprotein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, andother BLAST analyses provide further corroborative evidence that SEQ IDNO:2 is a KRAB family zinc finger protein.

In an alternative example, SEQ ID NO:16 is 93% identical, from residueMl to residue R364, to chicken transcription factor, LEF-l (GenBank IDg3258665) as determined by the Basic Local Alignment Search Tool(BLAST). (See Table 2.) The BLAST probability score is 9.8e-191, whichindicates the probability of obtaining the observed polypeptide sequencealignment by chance. SEQ ID NO:16 is localized to the nucleus, functionsas a DNA-binding protein, and is a transcriptional activator, asdetermined by BLAST analysis using the PROTEOME database. SEQ ID NO:16also contains a HMG (high mobility group) box domain as determined bysearching for statistically significant matches in the hidden Markovmodel (HMM)-based PFAM database of conserved protein family domains.(See Table 3.) Data from BLIMPS, and other BLAST analyses providefurther corroborative evidence that SEQ ID NO:16 is a LEF-1transcription factor.

In an alternative example, SEQ ID NO:19 is 71% identical from residueH19 to residue A1 13, and 100% identical from residue Ml to residue Y48,to ribosomal protein L27a (GenBank ID g550⁰¹7) as determined by theBasic Local Alignment Search Tool (BLAST). (See Table 2.) The BLASTprobability score is 1.4e-3 1, which indicates the probability ofobtaining the observed polypeptide sequence alignment by chance. SEQ IDNO:19 is a component of the large 60S ribosomal subunit, and isabnormally expressed in colorectal carcinomas, as determined by BLASTanalysis using the PROTEOME database. SEQ ID NO:19 also contains aribosomal protein L15 domain as determined by searching forstatistically significant matches in the hidden Markov model (HMM)-basedPFAM database of conserved protein family domains. (See Table 3.) Datafrom BLIMPS, MOTIFS, and PROFILESCAN analyses provide furthercorroborative evidence that SEQ ID NO:19 is a ribosomal protein.

In an alternative example, SEQ ID NO:51 is 98% identical, from residueMI to residue H477, to a human transcription factor (GenBank ID g516381)as determined by the Basic Local Alignment Search Tool (BLAST). (SeeTable 2.) The BLAST probability score is 3.5e-266, which indicates theprobability of obtaining the observed polypeptide sequence alignment bychance. SEQ ID NO:5 1 also has homology to proteins that are localizedto the neuronal cells, have DNA-binding and transcriptional regulationfunction, and are fork head proteins, as determined by BLAST analysisusing the PROTEOME database. SEQ ID NO:51 also contains a fork headdomain as determined by searching for statistically significant matchesin the hidden Markov model (HMM)-based PFAM database of conservedprotein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, andPROFHLESCAN analyses provide further corroborative evidence that SEQ IDNO:51 is a fork head DNA-binding protein.

SEQ ID NO:1, SEQ ID NO:3-15, SEQ ID NO:17-18, SEQ ID NO:20-50, and SEQID NO:52-58 were analyzed and annotated in a similar manner. Thealgorithms and parameters for the analysis of SEQ ID NO:1-58 aredescribed in Table 7.

As shown in Table 4, the full length polynucleotide embodiments wereassembled using cDNA sequences or coding (exon) sequences derived fromgenomic DNA, or any combination of these two types of sequences. Column1 lists the polynucleotide sequence identification number(Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotideconsensus sequence number (Incyte ID) for each polynucleotide of theinvention, and the length of each polynucleotide sequence in basepairs.Column 2 shows the nucleotide start (5′) and stop (3′) positions of thecDNA and/or genomic sequences used to assemble the full lengthpolynucleotide embodiments, and of fragments of the polynucleotideswhich are useful, for example, in hybridization or amplificationtechnologies that identify SEQ ID NO:59-116 or that distinguish betweenSEQ ID NO:59-116 and related polynucleotides.

The polynucleotide fragments described in Column 2 of Table 4 may referspecifically, for example, to Incyte cDNAs derived from tissue-specificcDNA libraries or from pooled cDNA libraries. Alternatively, thepolynucleotide fragments described in column 2 may refer to GenBankcDNAs or ESTs which contributed to the assembly of the full lengthpolynucleotides. In addition, the polynucleotide fragments described incolumn 2 may identify sequences derived from the ENSEMBL (The SangerCentre, Cambridge, UK) database (i.e., those sequences including thedesignation “ENST”). Alternatively, the polynucleotide fragmentsdescribed in column 2 may be derived from the NCBI RefSeq NucleotideSequence Records Database (i.e., those sequences including thedesignation “NM” or “NT”) or the NCBI RefSeq Protein Sequence Records(i. e., those sequences including the designation “NP”). Alternatively,the polynucleotide fragments described in column 2 may refer toassemblages of both cDNA and Genscan-predicted exons brought together byan “exon stitching” algorithm. For example, a polynucleotide sequenceidentified as FL_XXXKKK_N₁ _(—) N₂ _(—) YYYYY_N₃ _(—) N₄ represents a“stitched” sequence in which XXXXXX is the identification number of thecluster of sequences to which the algorithm was applied, and YYYYY isthe number of the prediction generated by the algorithm, andN_(1,2,3 . . .) , if present, represent specific exons that may havebeen manually edited during analysis (See Example V). Alternatively, thepolynucleotide fragments in column 2 may refer to assemblages of exonsbrought together by an “exon-stretching” algorithm. For example, apolynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_(—)1_N is a“stretched” sequence, with XXXXXX being the Incyte projectidentification number, gAAAAA being the GenBank identification number ofthe human genomic sequence to which the “exon-stretching” algorithm wasapplied, gBBBBB being the GenBank identification number or NCBI RefSeqidentification number of the nearest GenBank protein homolog, and Nreferring to specific exons (See Example V). In instances where a RefSeqsequence was used as a protein homolog for the “exon-stretching”algorithm, a RefSeq identifier (denoted by “NM,” “NP,” or “NT”) maybeused in place of the GenBank identifier (i.e., gBBBBB).

Alternatively, a prefix identifies component sequences that werehand-edited, predicted from genomic DNA sequences, or derived from acombination of sequence analysis methods. The following Table listsexamples of component sequence prefixes and corresponding sequenceanalysis methods associated with the prefixes (see Example IV andExample V). Prefix Type of analysis and/or examples of programs GNN,GFG, Exon prediction from genomic sequences using, for ENST example,GENSCAN (Stanford University, CA, USA) or FGENES (Computer GenomicsGroup, The Sanger Centre, Cambridge, UK). GBI Hand-edited analysis ofgenomic sequences. FL Stitched or stretched genomic sequences (seeExample V). INCY Full length transcript and exon prediction from mappingof EST sequences to the genome. Genomic location and EST compositiondata are combined to predict the exons and resulting transcript.

In some cases, Incyte cDNA coverage redundant with the sequence coverageshown in Table 4 was obtained to confirm the final consensuspolynucleotide sequence, but the relevant Incyte cDNA identificationnumbers are not shown.

Table 5 shows the representative cDNA libraries for those full lengthpolynucleotides which were assembled using Incyte cDNA sequences. Therepresentative cDNA library is the Incyte cDNA library which is mostfrequently represented by the Incyte cDNA sequences which were used toassemble and confirm the above polynucleotides. The tissues and vectorswhich were used to construct the cDNA libraries shown in Table 5 aredescribed in Table 6.

The invention also encompasses NAAP variants. Various embodiments ofNAAP variants can have at least about 80%, at least about 90%, or atleast about 95% amino acid sequence identity to the NAAP amino acidsequence, and can contain at least one functional or structuralcharacteristic of NAAP.

Various embodiments also encompass polynucleotides which encode NAAP. Ina particular embodiment, the invention encompasses a polynucleotidesequence comprising a sequence selected from the group consisting of SEQID NO:59-116, which encodes NAAP. The polynucleotide sequences of SEQ IDNO:59-116, as presented in the Sequence Listing, embrace the equivalentRNA sequences, wherein occurrences of the nitrogenous base thymine arereplaced with uracil, and the sugar backbone is composed of riboseinstead of deoxyribose.

The invention also encompasses variants of a polynucleotide encodingNAAP. In particular, such a variant polynucleotide will have at leastabout 70%, or alternatively at least about 85%, or even at least about95% polynucleotide sequence identity to a polynucleotide encoding NAAP.A particular aspect of the invention encompasses a variant of apolynucleotide comprising a sequence selected from the group consistingof SEQ ID NO:59-116 which has at least about 70%, or alternatively atleast about 85%, or even at least about 95% polynucleotide sequenceidentity to a nucleic acid sequence selected from the group consistingof SEQ ID NO:59-116. Any one of the polynucleotide variants describedabove can encode a polypeptide which contains at least one functional orstructural characteristic of NAAP.

In addition, or in the alternative, a polynucleotide variant of theinvention is a splice variant of a polynucleotide encoding NAAP. Asplice variant may have portions which have significant sequenceidentity to a polynucleotide encoding NAAP, but will generally have agreater or lesser number of polynucleotides due to additions ordeletions of blocks of sequence arising from alternate splicing of exonsduring mRNA processing. A splice variant may have less than about 70%,or alternatively less than about 60%, or alternatively less than about50% polynucleotide sequence identity to a polynucleotide encoding NAAPover its entire length; however, portions of the splice variant willhave at least about 70%, or alternatively at least about 85%, oralternatively at least about 95%, or alternatively 100% polynucleotidesequence identity to portions of the polynucleotide encoding NAAP. Forexample, a polynucleotide comprising a sequence of SEQ ID NO:105 and apolynucleotide comprising a sequence of SEQ ID NO:110 are splicevariants of each other. Any one of the splice variants described abovecan encode a polypeptide which contains at least one functional orstructural characteristic of NAAP.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding NAAP, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring NAAP, and all suchvariations are to be considered as being specifically disclosed.

Although polynucleotides which encode NAAP and its variants aregenerally capable of hybridizing to polynucleotides encoding naturallyoccurring NAAP under appropriately selected conditions of stringency, itmay be advantageous to produce polynucleotides encoding NAAP or itsderivatives possessing a substantially different codon usage, e.g.,inclusion of non-naturally occurring codons. Codons may be selected toincrease the rate at which expression of the peptide occurs in aparticular prokaryotic or eukaryotic host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encoding NAAPand its derivatives without altering the encoded amino acid sequencesinclude the production of RNA transcripts having more desirableproperties, such as a greater half-life, than transcripts produced fromthe naturally occurring sequence.

The invention also encompasses production of polynucleotides whichencode NAAP and NAAP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic polynucleotide maybe inserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a polynucleotideencoding NAAP or any fragment thereof.

Embodiments of the invention can also include polynucleotides that arecapable of hybridizing to the claimed polynucleotides, and, inparticular, to those having the sequences shown in SEQ ID NO:59-116 andfragments thereof, under various conditions of stringency (Wahl, G. M.and S. L. Berger (1987) Methods Enzymol. 152:399407; Kimmel, A. R.(1987) Methods Enzymol. 152:507-511). Hybridization conditions,including annealing and wash conditions, are described in “Definitions.”

Methods for DNA sequencing are well known in the art and may be used topractice any of the embodiments of the invention. The methods may employsuch enzymes as the Klenow fragment of DNA polymerase 1, SEQUENASE (USBiochemical, Cleveland Ohio), Taq polymerase (Applied Biosystems),thermostable T7 polymerase (Amersham Biosciences, Piscataway N.J.), orcombinations of polymerases and proofreading exonucleases such as thosefound in the ELONGASE amplification system (Invitrogen, CarlsbadCalif.). Preferably, sequence preparation is automated with machinessuch as the MICROLAB 2200 liquid transfer system (Hamilton, Reno Nev.),PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI CATALYST800 thermal cycler (Applied Biosystems). Sequencing is then carried outusing either the ABI 373 or 377 DNA sequencing system (AppliedBiosystems), the MEGABACE 1000 DNA sequencing system (AmershamBiosciences), or other systems known in the art. The resulting sequencesare analyzed using a variety of algorithms which are well known in theart (Ausubel et al., supra, ch. 7; Meyers, R. A. (1995) MolecularBiology and Biotechnology, Wiley V C H, New York N.Y., pp. 856-853).

The nucleic acids encoding NAAP may be extended utilizing a partialnucleotide sequence and employing various PCR-based methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector (Sarkar, G.(1993) PCR Methods Applic. 2:318-322). Another method, inverse PCR, usesprimers that extend in divergent directions to amplify unknown sequencefrom a circularized template. The template is derived from restrictionfragments comprising a known genomic locus and surrounding sequences(Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186). A third method,capture PCR, involves PCR amplification of DNA fragments adjacent toknown sequences in human and yeast artificial chromosome DNA(Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119). In thismethod, multiple restriction enzyme digestions and ligations may be usedto insert an engineered double-stranded sequence into a region ofunknown sequence before performing PCR. Other methods which may be usedto retrieve unknown sequences are known in the art (Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers maybe designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

When screening for full length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5′regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5′ non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, AppliedBiosystems), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotides or fragmentsthereof which encode NAAP may be cloned in recombinant DNA moleculesthat direct expression of NAAP, or fragments or functional equivalentsthereof, in appropriate host cells. Due to the inherent degeneracy ofthe genetic code, other polynucleotides which encode substantially thesame or a functionally equivalent polypeptides may be produced and usedto express NAAP.

The polynucleotides of the invention can be engineered using methodsgenerally known in the art in order to alter NAAP-encoding sequences fora variety of purposes including, but not limited to, modification of thecloning, processing, and/or expression of the gene product. DNAshuffling by random fragmentation and PCR reassembly of gene fragmentsand synthetic oligonucleotides may be used to engineer the nucleotidesequences. For example, oligonucleotide-mediated site-directedmutagenesis may be used to introduce mutations that create newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

The nucleotides of the present invention may be subjected to DNAshuffling techniques such as MOLECULARBREEDING (Maxygen Inc., SantaClara Calif.; described in U.S. Pat. No. 5,837,458; Chang, C.-C. et al.(1999) Nat. Biotechnol. 17:793-797; Christians, F. C. et al. (1999) Nat.Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol.14:315-319) to alter or improve the biological properties of NAAP, suchas its biological or enzymatic activity or its ability to bind to othermolecules or compounds. DNA shuffling is a process by which a library ofgene variants is produced using PCR-mediated recombination of genefragments. The library is then subjected to selection or screeningprocedures that identify those gene variants with the desiredproperties. These preferred variants may then be pooled and furthersubjected to recursive rounds of DNA shuffling and selection/screening.Thus, genetic diversity is created through “artificial” breeding andrapid molecular evolution. For example, fragments of a single genecontaining random point mutations may be recombined, screened, and thenreshuffled until the desired properties are optimized. Alternatively,fragments of a given gene may be recombined with fragments of homologousgenes in the same gene family, either from the same or differentspecies, thereby maximizing the genetic diversity of multiple naturallyoccurring genes in a directed and controllable manner.

In another embodiment, polynucleotides encoding NAAP may be synthesized,in whole or in part, using one or more chemical methods well known inthe art (Caruthers, M. H. et al. (1980) Nucleic Acids Symp. Ser.7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).Alternatively, NAAP itself or a fragment thereof may be synthesizedusing chemical methods known in the art. For example, peptide synthesiscan be performed using various solution-phase or solid-phase techniques(Creighton, T. (1984) Proteins, Structures and Molecular Properties, W HFreeman, New York N.Y., pp. 55-60; Roberge, J. Y. et al. (1995) Science269:202-204). Automated synthesis may be achieved using the ABI 43 1Apeptide synthesizer (Applied Biosystems). Additionally, the amino acidsequence of NAAP, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide or a polypeptide having asequence of a naturally occurring polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography (Chiez, R. M. and F. Z. Regnier (1990)Methods Enzymol. 182:392-421). The composition of the synthetic peptidesmay be confirmed by amino acid analysis or by sequencing (Creighton,supra, pp. 28-53).

In order to express a biologically active NAAP, the polynucleotidesencoding NAAP or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5′ and 3′ untranslated regions in the vector and in polynucleotidesencoding NAAP. Such elements may vary in their strength and specificity.Specific initiation signals may also be used to achieve more efficienttranslation of polynucleotides encoding NAAP. Such signals include theATG initiation codon and adjacent sequences, e.g. the Kozak sequence. Incases where a polynucleotide sequence encoding NAAP and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used (Scharf,D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing polynucleotides encoding NAAPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination (Sambrook and Russell,supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).

A variety of expression vector/host systems may be utilized to containand express polynucleotides encoding NAAP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrookand Russell, supra; Ausubel et al., supra; Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509; Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; TheMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. USA 81:3655-3659; Harrington, J. J. et al. (1997) Nat. Genet.15:345-355). Expression vectors derived from retroviruses, adenoviruses,or herpes or vaccinia viruses, or from various bacterial plasmids, maybe used for delivery of polynucleotides to the targeted organ, tissue,or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther.5:350-356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344;Buller, R. M. et al. (1985) Nature 317:813-815; McGregor, D. P. et al.(1994) Mol. Immunol. 31:219-226; Verma, I. M. and N. Somia (1997) Nature389:239-242). The invention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotides encodingNAAP. For example, routine cloning, subcloning, and propagation ofpolynucleotides encoding NAAP can be achieved using a multifunctional E.coli vector such as PBLUESCRRPT (Stratagene, La Jolla Calif.) or PSPORT1plasmid (Invitrogen). Ligation of polynucleotides encoding NAAP into thevector's multiple cloning site disrupts the lacZ gene, allowing acalorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509). When large quantities of NAAP are needed, e.g. for theproduction of antibodies, vectors which direct high level expression ofNAAP may be used. For example, vectors containing the strong, inducibleSP6 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of NAAP. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH promoters, may be used in the yeastSaccharomyces cerevisiae or Pichia pastoris. In addition, such vectorsdirect either the secretion or intracellular retention of expressedproteins and enable integration of foreign polynucleotide sequences intothe host genome for stable propagation (Ausubel et al., supra; Bitter,G. A. et al. (1987) Methods Enzymol. 153:516-544; Scorer, C. A. et al.(1994) Bio/Technology 12:181-184).

Plant systems may also be used for expression of NAAP. Transcription ofpolynucleotides encoding NAAP may be driven by viral promoters, e.g.,the 35S and 19S promoters of CaMV used alone or in combination with theomega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J.et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructscan be introduced into plant cells by direct DNA transformation orpathogen-mediated transfection (The McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York N.Y., pp. 191-196).

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,polynucleotides encoding NAAP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses NAAP in host cells (Logan, J. and T. Shenk (1984) Proc. Natl.Acad. Sci. USA 81:3655-3659). In addition, transcription enhancers, suchas the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells. SV40 or EBV-based vectors may alsobe used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes (Harrington, J. J. et al. (1997)Nat. Genet. 15:345-355).

For long term production of recombinant proteins in mammalian systems,stable expression of NAAP in cell lines is preferred. For example,polynucleotides encoding NAAP can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ and apr⁻ cells, respectively (Wigler, M. et al. (1977) Cell11:223-232; Lowy, I. et al. (1980) Cell 22:817-823). Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the arninoglycosides neomycinand G-418; and als and pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Wigler, M. et al.(1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-Garapin, F. etal. (1981) J. Mol. Biol. 150:1-14). Additional selectable genes havebeen described, e.g., trpB and hisD, which alter cellular requirementsfor metabolites (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.Acad. Sci. USA 85:8047-8051). Visible markers, e.g., anthocyanins, greenfluorescent proteins (GFP; Clontech), β-glucuronidase and its substrateβ-glucuronide, or luciferase and its substrate luciferin may be used.These markers can be used not only to identify transformants, but alsoto quantify the amount of transient or stable protein expressionattributable to a specific vector system (Rhodes, C. A. (1995) MethodsMol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingNAAP is inserted within a marker gene sequence, transformed cellscontaining polynucleotides encoding NAAP can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding NAAP under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

In general, host cells that contain the polynucleotide encoding NAAP andthat express NAAP may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of NAAPusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on NAAPis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art (Hampton, R. et al. (1990)Serological Methods, a Laboratory Manual, APS Press, St. Paul Minn.,Sect. IV; Coligan, J. E. et al. (1997) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York N.Y.; Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa N.J.).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NAAP includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, polynucleotides encodingNAAP, or any fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Biosciences, Promega (Madison Wis.), and US Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

Host cells transformed with polynucleotides encoding NAAP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNAAP may be designed to contain signal sequences which direct secretionof NAAP through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted polynucleotides or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” or “pro” form ofthe protein may also be used to specify protein targeting, folding,and/or activity. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available fromthe American Type Culture Collection (ATCC, Manassas Va.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding NAAP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric NAAPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of NAAP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the NAAP encodingsequence and the heterologous protein sequence, so that NAAP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel et al. (supra, ch. 10 and 16). A variety of commerciallyavailable kits may also be used to facilitate expression andpurification of fusion proteins.

In another embodiment, synthesis of radiolabeled NAAP may be achieved invitro using the TNT rabbit reticulocyte lysate or wheat germ extractsystem (Promega). These systems couple transcription and translation ofprotein-coding sequences operably associated with the T7, T3, or SP6promoters. Translation takes place in the presence of a radiolabeledamino acid precursor, for example, ³⁵S-methionine.

NAAP, fragments of NAAP, or variants of NAAP may be used to screen forcompounds that specifically bind to NAAP. One or more test compounds maybe screened for specific binding to NAAP. In various embodiments, 1, 2,3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened forspecific binding to NAAP. Examples of test compounds can includeantibodies, anticalins, oligonucleotides, proteins (e.g., ligands orreceptors), or small molecules.

In related embodiments, variants of NAAP can be used to screen forbinding of test compounds, such as antibodies, to NAAP, a variant ofNAAP, or a combination of NAAP and/or one or more variants NAAP. In anembodiment, a variant of NAAP can be used to screen for compounds thatbind to a variant of NAAP, but not to NAAP having the exact sequence ofa sequence of SEQ ID NO:1-58. NAAP variants used to perform suchscreening can have a range of about 50% to about 99% sequence identityto NAAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%,and 95% sequence identity.

In an embodiment, a compound identified in a screen for specific bindingto NAAP can be closely related to the natural ligand of NAAP, e.g., aligand or fragment thereof, a natural substrate, a structural orfunctional miinetic, or a natural binding partner (Coligan, J. E. et al.(1991) Current Protocols in Immunolor 1(2):Chapter 5). In anotherembodiment, the compound thus identified can be a natural ligand of areceptor NAAP (Howard, A. D. et al. (2001) Trends Pharmacol.Sci.22:132-140; Wise, A. et al. (2002) Drug Discovery Today 7:235-246).

In other embodiments, a compound identified in a screen for specificbinding to NAAP can be closely related to the natural receptor to whichNAAP binds, at least a fragment of the receptor, or a fragment of thereceptor including all or a portion of the ligand binding site orbinding pocket. For example, the compound may be a receptor for NAAPwhich is capable of propagating a signal, or a decoy receptor for NAAPwhich is not capable of propagating a signal (Ashkenazi, A. and V. M.Divit (1999) Curr. Opin. Cell Biol. 11:255-260; Mantovani, A. et al.(2001) Trends Imunol. 22:328-336). The compound can be rationallydesigned using known techniques. Examples of such techniques includethose used to construct the compound etanercept (ENBREL; Amgen Inc.,Thousand Oaks Calif.), which is efficacious for treating rheumatoidarthritis in humans. Etanercept is an engineered p75 tumor necrosisfactor (TNF) receptor dimer linked to the Fc portion of human IgG₁(Taylor, P. C. et al. (2001) Curr. Opin. Immunol. 13:611-616).

In one embodiment, two or more antibodies having similar or,alternatively, different specificities can be screened for specificbinding to NAAP, fragments of NAAP, or variants of NAAP. The bindingspecificity of the antibodies thus screened can thereby be selected toidentify particular fragments or variants of NAAP. In one embodiment, anantibody can be selected such that its binding specificity allows forpreferential identification of specific fragments or variants of NAAP.In another embodiment, an antibody can be selected such that its bindingspecificity allows for preferential diagnosis of a specific disease orcondition having increased, decreased, or otherwise abnormal productionof NAAP.

In an embodiment, anticalins can be screened for specific binding toNAAP, fragments of NAAP, or variants of NAAP. Anticalins areligand-binding proteins that have been constructed based on a lipocalinscaffold (Weiss, G. A. and H. B. Lowman (2000) Chem. Biol. 7:R177-R184;Skerra, A. (2001) J. Biotechnol. 74:257-275). The protein architectureof lipocalins can include a beta-barrel having eight antiparallelbeta-strands, which supports four loops at its open end. These loopsform the natural ligand-binding site of the lipocalins, a site which canbe re-engineered in vitro by amino acid substitutions to impart novelbinding specificities. The amino acid substitutions can be made usingmethods known in the art or described herein, and can includeconservative substitutions (e.g., substitutions that do not alterbinding specificity) or substitutions that modestly, moderately, orsignificantly alter binding specificity.

In one embodiment, screening for compounds which specifically bind to,stimulate, or inhibit NAAP involves producing appropriate cells whichexpress NAAP, either as a secreted protein or on the cell membrane.Preferred cells can include cells from mammals, yeast, Drosophila, or E.coli. Cells expressing NAAP or cell membrane fractions which containNAAP are then contacted with a test compound and binding, stimulation,or inhibition of activity of either NAAP or the compound is analyzed.

An assay may simply test binding of a test compound to the polypeptide,wherein binding is detected by a fluorophore, radioisotope, enzymeconjugate, or other detectable label. For example, the assay maycomprise the steps of combining at least one test compound with NAAP,either in solution or affixed to a solid support, and detecting thebinding of NAAP to the compound. Alternatively, the assay may detect ormeasure binding of a test compound in the presence of a labeledcompetitor. Additionally, the assay may be carried out using cell-freepreparations, chemical libraries, or natural product mixtures, and thetest compound(s) may be free in solution or affixed to a solid support.

An assay can be used to assess the ability of a compound to bind to itsnatural ligand and/or to inhibit the binding of its natural ligand toits natural receptors. Examples of such assays include radio-labelingassays such as those described in U.S. Pat. No. 5,914,236 and U.S. Pat.No. 6,372,724. In a related embodiment, one or more amino acidsubstitutions can be introduced into a polypeptide compound (such as areceptor) to improve or alter its ability to bind to its natural ligands(Matthews, D. J. and J. A. Wells. (1994) Chem. Biol. 1:25-30). Inanother related embodiment, one or more amino acid substitutions can beintroduced into a polypeptide compound (such as a ligand) to improve oralter its ability to bind to its natural receptors (Cunningham, B. C.and J. A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman,H. B. et al. (1991) J. Biol. Chem. 266:10982-10988).

NAAP, fragments of NAAP, or variants of NAAP may be used to screen forcompounds 1o that modulate the activity of NAAP. Such compounds mayinclude agonists, antagonists, or partial or inverse agonists. In oneembodiment, an assay is performed under conditions permissive for NAAPactivity, wherein NAAP is combined with at least one test compound, andthe activity of NAAP in the presence of a test compound is compared withthe activity of NAAP in the absence of the test compound. A change inthe activity of NAAP in the presence of the test compound is indicativeof a compound that modulates the activity of NAAP. Alternatively, a testcompound is combined with an in vitro or cell-free system comprisingNAAP under conditions suitable for NAAP activity, and the assay isperformed. In either of these assays, a test compound which modulatesthe activity of NAAP may do so indirectly and need not come in directcontact with the test compound. At least one and up to a plurality oftest compounds may be screened.

In another embodiment, polynucleotides encoding NAAP or their mammalianhomologs may be “knocked out” in an animal model system using homologousrecombination in embryonic stem (ES) cells. Such techniques are wellknown in the art and are useful for the generation of animal models ofhuman disease (see, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No.5,767,337). For example, mouse ES cells, such as the mouse 129/SvJ cellline, are derived from the early mouse embryo and grown in culture. TheES cells are transformed with a vector containing the gene of interestdisrupted by a marker gene, e.g., the neomycin phosphotransferase gene(neo; Capecchi, M. R. (1989) Science 244:1288-1292). The vectorintegrates into the corresponding region of the host genome byhomologous recombination. Alternatively, homologous recombination takesplace using the Cre-loxP system to knockout a gene of interest in atissue- or developmental stage-specific manner (Marth, J. D. (1996)Clin. Invest. 97:1999-2002; Wagner, K. U. et al. (1997) Nucleic AcidsRes. 25:4323-4330). Transformed ES cells are identified andmicroinjected into mouse cell blastocysts such as those from the C57BL/6mouse strain. The blastocysts are surgically transferred topseudopregnant dams, and the resulting chimeric progeny are genotypedand bred to produce heterozygous or homozygous strains. Transgenicanimals thus generated may be tested with potential therapeutic or toxicagents.

Polynucleotides encoding NAAP may also be manipulated in vitro in EScells derived from human blastocysts. Human ES cells have the potentialto differentiate into at least eight separate cell lineages includingendoderm, mesoderm, and ectodermal cell types. These cell lineagesdifferentiate into, for example, neural cells, hematopoietic lineages,and cardiomyocytes (Thomson, J. A. et al. (1998) Science 282:1145-1147).

Polynucleotides encoding NAAP can also be used to create “knockin”humanized animals (pigs) or transgenic animals (mice or rats) to modelhuman disease. With knockin technology, a region of a polynucleotideencoding NAAP is injected into animal ES cells, and the injectedsequence integrates into the animal cell genome. Transformed cells areinjected into blastulae, and the blastulae are implanted as describedabove. Transgenic progeny or inbred lines are studied and treated withpotential pharmaceutical agents to obtain information on treatment of ahuman disease. Alternatively, a mammal inbred to overexpress NAAP, e.g.,by secreting NAAP in its milk, may also serve as a convenient source ofthat protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74).

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between regions of NAAP and nucleic acid-associatedproteins. In addition, examples of tissues expressing NAAP can be foundin Table 6 and can also be found in Example XL. Therefore, NAAP appearsto play a role in cell proliferative, neurological, developmental, andautoimmune/inflammatory disorders, and infections. In the treatment ofdisorders associated with increased NAAP expression or activity, it isdesirable to decrease the expression or activity of NAAP. In thetreatment of disorders associated with decreased NAAP expression oractivity, it is desirable to increase the expression or activity ofNAAP.

Therefore, in one embodiment, NAAP or a fragment or derivative thereofmay be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of NAAP. Examples ofsuch disorders include, but are not limited to, a cell proliferativedisorder such as actinic keratosis, arteriosclerosis, atherosclerosis,bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD),myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera,psoriasis, primary thrombocythemia, and cancers includingadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, a cancer of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, colon, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a neurological disordersuch as epilepsy, ischemic cerebrovascular disease, stroke, cerebralneoplasms, Alzheimer's disease, Pick's disease, Huntington's disease,dementia, Parkinson's disease and other extrapyramidal disorders,amyotrophic lateral sclerosis and other motor neuron disorders,progressive neural muscular atrophy, retinitis pigmentosa, hereditaryataxias, multiple sclerosis and other demyelinating diseases, bacterialand viral meningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease, prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome,fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorder of the central nervous system, cerebralpalsy, a neuroskeletal disorder, an autonomic nervous system disorder, acranial nerve disorder, a spinal cord disease, muscular dystrophy andother neuromuscular disorder, a peripheral nervous system disorder,dermatomyositis and polymyositis, inherited, metabolic, endocrine, andtoxic myopathy, myasthenia gravis, periodic paralysis, a mental disorderincluding mood, anxiety, and schizophrenic disorder, seasonal affectivedisorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy,tardive dyskinesia, dystonias, paranoid psychoses, postherpeticneuralgia, and Tourette's disorder; a developmental disorder such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss; anautoimmune/inflammatory disorder such as acquired immunodeficiencysyndrome (AIDS), Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma; andan infection, such as those caused by a viral agent classified asadenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus,hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus,papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus,retrovirus, rhabdovirus, or togavirus; an infection caused by abacterial agent classified as pneumococcus, staphylococcus,streptococcus, bacillus, corynebacterium, clostridium, meningococcus,gonococcus, listeria, moraxella, kingella, haemophilus, legionella,bordetella, gram-negative enterobacterium including shigella,salmonella, or campylobacter, pseudomonas, vibrio, brucella,francisella, yersinia, bartonella, norcardium, actinomyces,mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; aninfection caused by a fungal agent classified as aspergillus,blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia,histoplasma, or other mycosis-causing fungal agent; and an infectioncaused by a parasite classified as plasmodium or malaria-causing,parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystiscarinii, intestinal protozoa such as giardia, trichomonas, tissuenematode such as trichinella, intestinal nematode such as ascaris,lymphatic filarial nematode, trematode such as schistosoma, and cestodesuch as tapeworm.

In another embodiment, a vector capable of expressing NAAP or a fragmentor derivative thereof may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofNAAP including, but not limited to, those described above.

In a further embodiment, a composition comprising a substantiallypurified NAAP in conjunction with a suitable pharmaceutical carrier maybe administered to a subject to treat or prevent a disorder associatedwith decreased expression or activity of NAAP including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofNAAP may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of NAAP including, butnot limited to, those listed above.

In a further embodiment, an antagonist of NAAP may be administered to asubject to treat or prevent a disorder associated with increasedexpression or activity of NAAP. Examples of such disorders include, butare not limited to, those cell proliferative, neurological,developmental, and autoinmuunefinflammatory disorders, and infectionsdescribed above. In one aspect, an antibody which specifically bindsNAAP may be used directly as an antagonist or indirectly as a targetingor delivery mechanism for bringing a pharmaceutical agent to cells ortissues which express NAAP.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding NAAP may be administered to a subject to treator prevent a disorder associated with increased expression or activityof NAAP including, but not limited to, those described above.

In other embodiments, any protein, agonist, antagonist, antibody,complementary sequence, or vector embodiments may be administered incombination with other appropriate therapeutic agents. Selection of theappropriate agents for use in combination therapy may be made by one ofordinary skill in the art, according to conventional pharmaceuticalprinciples. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of NAAP may be produced using methods which are generallyknown in the art. In particular, purified NAAP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind NAAP. Antibodies to NAAP may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. In an embodiment, neutralizing antibodies (i.e.,those which inhibit dimer formation) can be used therapeutically. Singlechain antibodies (e.g., from camels or llamas) may be potent enzymeinhibitors and may have application in the design of peptide mimetics,and in the development of immuno-adsorbents and biosensors (Muyldermans,S. (2001) J. Biotechnol. 74:277-302).

For the production of antibodies, various hosts including goats,rabbits, rats, mice, camels, dromedaries, llamas, humans, and others maybe immunized by injection with NAAP or with any fragment or oligopeptidethereof which has immunogenic properties. Depending on the host species,various adjuvants may be used to increase immunological response. Suchadjuvants include, but are not limited to, Freund's, mineral gels suchas aluminum hydroxide, and surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,KLH, and dinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to NAAP have an amino acid sequence consisting of atleast about 5 amino acids, and generally will consist of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are substantially identical to a portion of theamino acid sequence of the natural protein. Short stretches of NAAPamino acids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to NAAP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. CellBiol. 62:109-120).

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceNAAP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837;Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for NAAP mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)₂ fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity (Huse, W. D. etal. (1989) Science 246:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between NAAP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NAAP epitopes is generally used, but a competitivebinding assay may also be employed (Pound, supra).

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for NAAP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of NAAP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple NAAP epitopes, represents the average affinity,or avidity, of the antibodies for NAAP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular NAAP epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theNAAP-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of NAAP, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. and A.Cryer (1991) A Practical Guide to Monoclonal Antibodies, John Wiley &Sons, New York N.Y.).

The titer and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is generally employed in proceduresrequiring precipitation of NAAP-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable (Catty, supra; Coligan et al., supra).

In another embodiment of the invention, polynucleotides encoding NAAP,or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, modifications of gene expression can beachieved by designing complementary sequences or antisense molecules(DNA, RNA, PNA, or modified oligonucleotides) to the coding orregulatory regions of the gene encoding NAAP. Such technology is wellknown in the art, and antisense oligonucleotides or larger fragments canbe designed from various locations along the coding or control regionsof sequences encoding NAAP (Agrawal, S., ed. (1996) AntisenseTherapeutics, Humana Press, Totawa N.J.).

In therapeutic use, any gene delivery system suitable for introductionof the antisense sequences into appropriate target cells can be used.Antisense sequences can be delivered intracellularly in the form of anexpression plasmid which, upon transcription, produces a sequencecomplementary to at least a portion of the cellular sequence encodingthe target protein (Slater, J. E. et al. (1998) J. Allergy Clin.Immunol. 102:469-475; Scanlon, K. J. et al. (1995) 9:1288-1296).Antisense sequences can also be introduced intracellularly through theuse of viral vectors, such as retrovirus and adeno-associated virusvectors (Miller, A. D. (1990) Blood 76:271; Ausubel et al., supra;Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63:323-347). Othergene delivery mechanisms include liposome-derived systems, artificialviral envelopes, and other systems known in the art (Rossi, J. J. (1995)Br. Med. Bull. 51:217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.87:1308-1315; Morris, M. C. et al. (1997) Nucleic Acids Res.25:2730-2736).

In another embodiment of the invention, polynucleotides encoding NAAPmay be used for somatic or germline gene therapy. Gene therapy may beperformed to (i) correct a genetic deficiency (e.g., in the cases ofsevere combined immunodeficiency (SCID)-X1 disease characterized byX-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science288:669-672), severe combined immunodeficiency syndrome associated withan inherited adenosine deaminase (ADA) deficiency (Blaese, R. M. et al.(1995) Science 270:475-480; Bordignon, C. et al. (1995) Science270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216;Crystal, R. G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R. G.et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, familialhypercholesteroleria, and hemophilia resulting from Factor VIII orFactor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express aconditionally lethal gene product (e.g., in the case of cancers whichresult from unregulated cell proliferation), or (iii) express a proteinwhich affords protection against intracellular parasites (e.g., againsthuman retroviruses, such as human immunodeficiency virus (HI)(Baltimore, D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996)Proc. Natl. Acad. Sci. USA 93:11395-11399), hepatitis B or C virus (HBV,HCV); fungal parasites, such as Candida albicans and Paracoccidioidesbrasiliensis; and protozoan parasites such as Plasmodium falciparum andTrypanosoma cruzi). In the case where a genetic deficiency in NAAPexpression or regulation causes disease, the expression of NAAP from anappropriate population of transduced cells may alleviate the clinicalmanifestations caused by the genetic deficiency.

In a further embodiment of the invention, diseases or disorders causedby deficiencies in NAAP are treated by constructing mammalian expressionvectors encoding NAAP and introducing these vectors by mechanical meansinto NAAP-deficient cells. Mechanical transfer technologies for use withcells in vivo or ex vitro include (i) direct DNA microinjection intoindividual cells, (ii) ballistic gold particle delivery, (iii)liposome-mediated transfection, (iv) receptor-mediated gene transfer,and (v) the use of DNA transposons (Morgan, R. A. and W. F. Anderson(1993) Annu. Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510;Boulay, J.-L. and H. Récipon (1998) Curr. Opin. Biotechnol. 9:445-450).

Expression vectors that may be effective for the expression of NAAPinclude, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP,PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT,PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.), and PTET-OFF,PTET-ON, PTRE2, NTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). NAAPmaybe expressed using (i) a constitutively active promoter, (e.g., fromcytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidinekinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., thetetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr. Opin.Biotechnol. 9:451-456), commercially available in the T-REX plasmid(Invitrogen)); the ecdysone-inducible promoter (available in theplasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin induciblepromoter; or the RU486/mifepristone inducible promoter (Rossi, F. M. V.and H. M. Blau, supra)), or (iii) a tissue-specific promoter or thenative promoter of the endogenous gene encoding NAAP from a normalindividual.

Commercially available liposome transformation kits (e.g., the PERFECTLIPID TRANSFECTION KIT, available from Invitrogen) allow one withordinary skill in the art to deliver polynucleotides to target cells inculture and require minimal effort to optimize experimental parameters.In the alternative, transformation is performed using the calciumphosphate method (Graham, F. L. and A. J. Eb (1973) Virology52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J.1:841-845). The introduction of DNA to primary cells requiresmodification of these standardized mammalian transfection protocols.

In another embodiment of the invention, diseases or disorders caused bygenetic defects with respect to NAAP expression are treated byconstructing a retrovirus vector consisting of (i) the polynucleotideencoding NAAP under the control of an independent promoter or theretrovirus long terminal repeat (LTR) promoter, (ii) appropriate RNApackaging signals, and (iii) a Rev-responsive element (RRE) along withadditional retrovirus cis-acting RNA sequences and coding sequencesrequired for efficient vector propagation. Retrovirus vectors (e.g., PFBand PFBNEO) are commercially available (Stratagene) and are based onpublished data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA92:6733-6737), incorporated by reference herein. The vector ispropagated in an appropriate vector producing cell line (VPCL) thatexpresses an envelope gene with a tropism for receptors on the targetcells or a promiscuous envelope protein such as VSVg (Armentano, D. etal. (1987) J. Virol. 61:1647-1650; Bender, M. A. et al. (1987) J. Virol.61:1639-1646; Adam, M. A. and A. D. Miller (1988) J. Virol.62:3802-3806; Dull, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey,R. et al. (1998) J. Virol. 72:9873-9880). U.S. Pat. No. 5,910,⁴³⁴ toRigg (“Method for obtaining retrovinis packaging cell lines producinghigh transducing efficiency retroviral supernatant”) discloses a methodfor obtaining retrovirus packaging cell lines and is hereby incorporatedby reference. Propagation of retrovirus vectors, transduction of apopulation of cells (e.g., CD4⁺ T-cells), and the return of transducedcells to a patient are procedures well known to persons skilled in theart of gene therapy and have been well documented (Ranga, U. et al.(1997) J. Virol. 71:7020-7029; Bauer, G. et al. (1997) Blood89:2259-2267; Bonyhadi, M. L. (1997) J. Virol. 71:4707-4716; Ranga, U.et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)Blood 89:2283-2290).

In an embodiment, an adenovirus-based gene therapy delivery system isused to deliver polynucleotides encoding NAAP to cells which have one ormore genetic abnormalities with respect to the expression of NAAP. Theconstruction and packaging of adenovirus-based vectors are well known tothose with ordinary skill in the art. Replication defective adenovirusvectors have proven to be versatile for importing genes encodingimmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially usefuladenoviral vectors are described in U.S. Pat. No. 5,707,618 to Armentano(“Adenovirus vectors for gene therapy”), hereby incorporated byreference. For adenoviral vectors, see also Antinozzi, P. A. et al.(1999; Annu. Rev. Nutr. 19:511-544) and Verma, I. M. and N. Somia (1997;Nature 18:389:239-242).

In another embodiment, a herpes-based, gene therapy delivery system isused to deliver polynucleotides encoding NAAP to target cells which haveone or more genetic abnormalities with respect to the expression ofNAAP. The use of herpes simplex virus (HSV)-based vectors may beespecially valuable for introducing NAAP to cells of the central nervoussystem, for which HSV has a tropism. The construction and packaging ofherpes-based vectors are well known to those with ordinary skill in theart. A replication-competent herpes simplex virus (HSV) type 1-basedvector has been used to deliver a reporter gene to the eyes of primates(Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The construction of aHSV-1 virus vector has also been disclosed in detail in U.S. Pat. No.5,804,413 to DeLuca (“Herpes simplex virus strains for gene transfer”),which is hereby incorporated by reference. U.S. Pat. No. 5,804,413teaches the use of recombinant HSV d92 which consists of a genomecontaining at least one exogenous gene to be transferred to a cell underthe control of the appropriate promoter for purposes including humangene therapy. Also taught by this patent are the construction and use ofrecombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSVvectors, see also Goins, W. F. et al. (1999; J. Virol. 73:519-532) andXu, H. et al. (1994; Dev. Biol. 163:152-161). The manipulation of clonedherpesvirus sequences, the generation of recombinant virus following thetransfection of multiple plasmids containing different segments of thelarge herpesvirus genomes, the growth and propagation of herpesvirus,and the infection of cells with herpesvirus are techniques well known tothose of ordinary skill in the art.

In another embodiment, an alphavirus (positive, single-stranded RNAvirus) vector is used to deliver polynucleotides encoding NAAP to targetcells. The biology of the prototypic alphavirus, Semliki Forest Virus(SFV), has been studied extensively and gene transfer vectors have beenbased on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin.Biotechnol. 9:464-469). During alphavirus RNA replication, a subgenornicRNA is generated that normally encodes the viral capsid proteins. Thissubgenomic RNA replicates to higher levels than the full length genoricRNA, resulting in the overproduction of capsid proteins relative to theviral proteins with enzymatic activity (e.g., protease and polymerase).Similarly, inserting the coding sequence for NAAP into the alphavirusgenome in place of the capsid-coding region results in the production ofa large number of NAAP-coding RNAs and the synthesis of high levels ofNAAP in vector transduced cells. While alphavirus infection is typicallyassociated with cell lysis within a few days, the ability to establish apersistent infection in hamster normal kidney cells (BHK-21) with avariant of Sindbis virus (SIN) indicates that the lytic replication ofalphaviruses can be altered to suit the needs of the gene therapyapplication (Dryga, S. A. et al. (1997) Virology 228:74-83). The widehost range of alphaviruses will allow the introduction of NAAP into avariety of cell types. The specific transduction of a subset of cells ina population may require the sorting of cells prior to transduction. Themethods of manipulating infectious cDNA clones of alphaviruses,performing alphavirus cDNA and RNA transfections, and performingalphavirus infections, are well known to those with ordinary skill inthe art.

Oligonucleotides derived from the transcription initiation site, e.g.,between about positions −10 and +10 from the start site, may also beemployed to inhibit gene expression. Similarly, inhibition can beachieved using triple helix base-pairing methodology. Triple helixpairing is useful because it causes inhibition of the ability of thedouble helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) in Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp. 163-177).A complementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of RNA moleculesencoding NAAP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes may be preparedby any method known in the art for the synthesis of nucleic acidmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA molecules encoding NAAP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize complementary RNA, constitutively or inducibly, can beintroduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3 ′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

In other embodiments of the invention, the expression of one or moreselected polynucleotides of the present invention can be altered,inhibited, decreased, or silenced using RNA interference (RNAi) orpost-transcriptional gene silencing (PTGS) methods known in the art.RNAi is a post-transcriptional mode of gene silencing in whichdouble-stranded RNA (dsRNA) introduced into a targeted cell specificallysuppresses the expression of the homologous gene (i.e., the gene bearingthe sequence complementary to the dsRNA). This effectively knocks out orsubstantially reduces the expression of the targeted gene. PIGS can alsobe accomplished by use of DNA or DNA fragments as well. RNAi methods aredescribed by Fire, A. et al. (1998; Nature 391:806-811) and Gura, T.(2000; Nature 404:804-808). PTGS can also be initiated by introductionof a complementary segment of DNA into the selected tissue using genedelivery and/or viral vector delivery methods described herein or knownin the art.

RNAi can be induced in mammalian cells by the use of small interferingRNA also known as siRNA. SiRNA are shorter segments of dsRNA (typicallyabout 21 to 23 nucleotides in length) that result in vivo from cleavageof introduced dsRNA by the action of an endogenous ribonuclease. SiRNAappear to be the mediators of the RNAi effect in mammals. The mosteffective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3′overhangs. The use of siRNA for inducing RNAi in mammalian cells isdescribed by Elbashir, S. M. et al. (2001; Nature 411:494-498).

SiRNA can either be generated indirectly by introduction of dsRNA intothe targeted cell, or directly by mammalian transfection methods andagents described herein or known in the art (such as liposome-mediatedtransfection, viral vector methods, or other polynucleotidedelivery/introductory methods). Suitable SiRNAs can be selected byexamining a transcript of the target polynucleotide (e.g., mRNA) fornucleotide sequences downstream from the AUG start codon and recordingthe occurrence of each nucleotide and the 3′ adjacent 19 to 23nucleotides as potential siRNA target sites, with sequences having a 21nucleotide length being preferred. Regions to be avoided for targetsiRNA sites include the 5′ and 3′ untranslated regions (UTRs) andregions near the start codon (within 75 bases), as these may be richerin regulatory protein binding sites. UTR-binding proteins and/ortranslation initiation complexes may interfere with binding of the siRNPendonuclease complex. The selected target sites for siRNA can then becompared to the appropriate genome database (e.g., human, etc.) usingBLAST or other sequence comparison algorithms known in the art. Targetsequences with significant homology to other coding sequences can beeliminated from consideration. The selected SiRNAs can be produced bychemical synthesis methods known in the art or by in vitro transcriptionusing commercially available methods and kits such as the SILENCER siRNAconstruction kit (Ambion, Austin Tex.).

In alternative embodiments, long-term gene silencing and/or RNAi effectscan be induced in selected tissue using expression vectors thatcontinuously express siRNA. This can be accomplished using expressionvectors that are engineered to express hairpin RNAs (shRNAs) usingmethods known in the art (see, e.g., Brummelkamp, T. R. et al. (2002)Science 296:550-553; and Paddison, P. J. et al. (2002) Genes Dev.16:948-958). In these and related embodiments, shRNAs can be deliveredto target cells using expression vectors known in the art. An example ofa suitable expression vector for delivery of siRNA is thePSILENCER1.0-U6 (circular) plasmid (Ambion). Once delivered to thetarget tissue, shRNAs are processed in vivo into siRNA-like moleculescapable of carrying out gene-specific silencing.

In various embodiments, the expression levels of genes targeted by RNAior PTGS methods can be determined by assays for mRNA and/or proteinanalysis. Expression levels of the mRNA of a targeted gene, can bedetermined by northern analysis methods using, for example, theNORTHERNMAX-GLY kit (Ambion); by microarray methods; by PCR methods; byreal time PCR methods; and by other RNA/polynucleotide assays known inthe art or described herein. Expression levels of the protein encoded bythe targeted gene can be determined by Western analysis using standardtechniques known in the art.

An additional embodiment of the invention encompasses a method forscreening for a compound which is effective in altering expression of apolynucleotide encoding NAAP. Compounds which may be effective inaltering expression of a specific polynucleotide may include, but arenot limited to, oligonucleotides, antisense oligonucleotides, triplehelix-forming oligonucleotides, transcription factors and otherpolypeptide transcriptional regulators, and non-macromolecular chemicalentities which are capable of interacting with specific polynucleotidesequences. Effective compounds may alter polynucleotide expression byacting as either inhibitors or promoters of polynucleotide expression.Thus, in the treatment of disorders associated with increased NAAPexpression or activity, a compound which specifically inhibitsexpression of the polynucleotide encoding NAAP may be therapeuticallyuseful, and in the treatment of disorders associated with decreased NAAPexpression or activity, a compound which specifically promotesexpression of the polynucleotide encoding NAAP may be therapeuticallyuseful.

In various embodiments, one or more test compounds may be screened foreffectiveness in altering expression of a specific polynucleotide. Atest compound may be obtained by any method commonly known in the art,including chemical modification of a compound known to be effective inaltering polynucleotide expression; selection from an existing,commercially-available or proprietary library of naturally-occurring ornon-natural chemical compounds; rational design of a compound based onchemical and/or structural properties of the target polynucleotide; andselection from a library of chemical compounds created combinatoriallyor randomly. A sample comprising a polynucleotide encoding NAAP isexposed to at least one test compound thus obtained. The sample maycomprise, for example, an intact or permeabilized cell, or an in vitrocell-free or reconstituted biochemical system. Alterations in theexpression of a polynucleotide encoding NAAP are assayed by any methodcommonly known in the art. Typically, the expression of a specificnucleotide is detected by hybridization with a probe having a nucleotidesequence complementary to the sequence of the polynucleotide encodingNAAP. The amount of hybridization may be quantified, thus forming thebasis for a comparison of the expression of the polynucleotide both withand without exposure to one or more test compounds. Detection of achange in the expression of a polynucleotide exposed to a test compoundindicates that the test compound is effective in altering the expressionof the polynucleotide. A screen for a compound effective in alteringexpression of a specific polynucleotide can be carried out, for example,using a Schizosaccharomyces pombe gene expression system (Atkins, D. etal. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et al. (2000) NucleicAcids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M. L.et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particularembodiment of the present invention involves screening a combinatoriallibrary of oligonucleotides (such as deoxyribonucleotides,ribonucleotides, peptide nucleic acids, and modified oligonucleotides)for antisense activity against a specific polynucleotide sequence(Bruice, T. W. et al. (1997) U.S. Pat. No. 5,686,242; Bruice, T. W. etal. (2000) U.S. Pat. No. 6,022,691).

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art (Goldman, C. K. et al. (1997) Nat. Biotechnol.15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such ashumans, dogs, cats, cows, horses, rabbits, and monkeys.

An additional embodiment of the invention relates to the administrationof a composition which generally comprises an active ingredientformulated with a pharmaceutically acceptable excipient. Excipients mayinclude, for example, sugars, starches, celluloses, gums, and proteins.Various formulations are commonly known and are thoroughly discussed inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.). Such compositions may consist of NAAP,antibodies to NAAP, and mimetics, agonists, antagonists, or inhibitorsof NAAP.

In various embodiments, the compositions described herein, such aspharmaceutical compositions, may be administered by any number of routesincluding, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal,enteral, topical, sublingual, or rectal means.

Compositions for pulmonary administration may be prepared in liquid ordry powder form. These compositions are generally aerosolizedimmediately prior to inhalation by the patient. In the case of smallmolecules (e.g. traditional low molecular weight organic drugs), aerosoldelivery of fast-acting formulations is well-known in the art. In thecase of macromolecules (e.g. larger peptides and proteins), recentdevelopments in the field of pulmonary delivery via the alveolar regionof the lung have enabled the practical delivery of drugs such as insulinto blood circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.5,997,848). Pulmonary delivery allows administration without needleinjection, and obviates the need for potentially toxic penetrationenhancers.

Compositions suitable for use in the invention include compositionswherein the active ingredients are contained in an effective amount toachieve the intended purpose. The determination of an effective dose iswell within the capability of those skilled in the art.

Specialized forms of compositions may be prepared for directintracellular delivery of macromolecules comprising NAAP or fragmentsthereof. For example, liposome preparations containing acell-impermeable macromolecule may promote cell fusion and intracellulardelivery of the macromolecule. Alternatively, NAAP or a fragment thereofmay be joined to a short cationic N-terminal portion from the HIV Tat-1protein. Fusion proteins thus generated have been found to transduceinto the cells of all tissues, including the brain, in a mouse modelsystem (Schwarze, S. R. et al. (1999) Science 285:1569-1572).

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models such as mice, rats, rabbits, dogs, monkeys, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example NAAP or fragments thereof, antibodies of NAAP,and agonists, antagonists or inibitors of NAAP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, which can be expressed asthe ID₅₀/ED₅₀ ratio. Compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies are used to formulate a range of dosage for human use.The dosage contained in such compositions is preferably within a rangeof circulating concentrations that includes the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting compositions may beadministered every 3 to 4 days, every week, or biweekly depending on thehalf-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind NAAP may beused for the diagnosis of disorders characterized by expression of NAAP,or in assays to monitor patients being treated with NAAP or agonists,antagonists, or inhibitors of NAAP. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for NAAP include methods which utilizethe antibody and a label to detect NAAP in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring NAAP, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of NAAP expression. Normal or standard values for NAAPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, for example, human subjects, withantibodies to NAAP under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, such as photometric means. Quantities of NAAP expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, polynucleotides encoding NAAPmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotides, complementary RNA and DNA molecules, andPNAs. The polynucleotides may be used to detect and quantify geneexpression in biopsied tissues in which expression of NAAP may becorrelated with disease. The diagnostic assay may be used to determineabsence, presence, and excess expression of NAAP, and to monitorregulation of NAAP levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotides, including genonic sequences, encoding NAAP orclosely related molecules may be used to identify nucleic acid sequenceswhich encode NAAP. The specificity of the probe, whether it is made froma highly specific region, e.g., the 5′ regulatory region, or from a lessspecific region, e.g., a conserved motif, and the stringency of thehybridization or amplification will determine whether the probeidentifies only naturally occurring sequences encoding NAAP, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, and mayhave at least 50% sequence identity to any of the NAAP encodingsequences. The hybridization probes of the subject invention may be DNAor RNA and may be derived from the sequence of SEQ ID NO:59-116 or fromgenomic sequences including promoters, enhancers, and introns of theNAAP gene.

Means for producing specific hybridization probes for polynucleotidesencoding NAAP include the cloning of polynucleotides encoding NAAP orNAAP derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotides encoding NAAP may be used for the diagnosis of disordersassociated with expression of NAAP. Examples of such disorders include,but are not limited to, a cell proliferative disorder such as actinickeratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,hepatitis, mixed connective tissue disease (MCID)), myelofibrosis,paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis,primary thrombocythemia, and cancers including adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, a cancer of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, colon, gall bladder, ganglia, gastrointestinaltract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid,penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid,and uterus; a neurological disorder such as epilepsy, ischemiccerebrovascular disease, stroke, cerebral neoplasms, Alzheimer'sdisease, Pick's disease, Huntington's disease, dementia, Parkinson'sdisease and other extrapyramidal disorders, amyotrophic lateralsclerosis and other motor neuron disorders, progressive neural muscularatrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosisand other demyelinating diseases, bacterial and viral meningitis, brainabscess, subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease, prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorder of the central nervous system, cerebralpalsy, a neuroskeletal disorder, an autonomic nervous system disorder, acranial nerve disorder, a spinal cord disease, muscular dystrophy andother neuromuscular disorder, a peripheral nervous system disorder,dermatomyositis and polymyositis, inherited, metabolic, endocrine, andtoxic myopathy, myasthenia gravis, periodic paralysis, a mental disorderincluding mood, anniety, and schizophrenic disorder, seasonal affectivedisorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy,tardive dyskinesia, dystonias, paranoid psychoses, postherpeticneuralgia, and Tourette's disorder; a developmental disorder such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss; anautoimmune/inflammatory disorder such as acquired immunodeficiencysyndrome (ADDS), Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthitis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helninthic infections, and trauma; andan infection, such as those caused by a viral agent classified asadenovirus, arenavirus, bunyavirus, calicivirus, coronavirus, filovirus,hepadnavirus, herpesvirus, flavivirus, orthomyxovirus, parvovirus,papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus,retrovirus, rhabdovirus, or togavirus; an infection caused by abacterial agent classified as pneumococcus, staphylococcus,streptococcus, bacillus, corynebacterium, clostridium, meningococcus,gonococcus, listeria, moraxella, kingella, haemophilus, legionella,bordetella, gram-negative enterobacterium including shigella,salmonella, or campylobacter, pseudomonas, vibrio, brucella,francisella, yersinia, bartonella, norcardium, actinomyces,mycobacterium, spirochaetale, rickettsia, chlamydia, or mycoplasma; aninfection caused by a fungal agent classified as aspergillus,blastomyces, dermatophytes, cryptococcus, coccidioides, malasezzia,histoplasma, or other mycosis-causing fungal agent; and an infectioncaused by a parasite classified as plasmodium or malaria-causing,parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystiscarinii, intestinal protozoa such as giardia, trichomonas, tissuenematode such as trichinella, intestinal nematode such as ascaris,lymphatic filarial nematode, trematode such as schistosoma, and cestodesuch as tapeworm. Polynucleotides encoding NAAP may be used in Southernor northern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and multiformat ELISA-like assays;and in microarrays utilizing fluids or tissues from patients to detectaltered NAAP expression. Such qualitative or quantitative methods arewell known in the art.

In a particular embodiment, polynucleotides encoding NAAP may be used inassays that detect the presence of associated disorders, particularlythose mentioned above. Polynucleotides complementary to sequencesencoding NAAP may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantified and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of polynucleotides encoding NAAP in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of NAAP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding NAAP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of an abnormal amount of transcript(either under- or overexpressed) in biopsied tissue from an individualmay indicate a predisposition for the development of the disease, or mayprovide a means for detecting the disease prior to the appearance ofactual clinical symptoms. A more definitive diagnosis of this type mayallow health professionals to employ preventative measures or aggressivetreatment earlier, thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding NAAP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding NAAP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding NAAP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantification of closely related DNA or RNA sequences.

In a particular aspect, oligonucleotide primers derived frompolynucleotides encoding NAAP may be used to detect single nucleotidepolymorphisms (SNPs). SNPs are substitutions, insertions and deletionsthat are a frequent cause of inherited or acquired genetic disease inhumans. Methods of SNP detection include, but are not limited to,single-stranded conformation polymorphism (SSCP) and fluorescent SSCP(fSSCP) methods. In SSCP, oligonucleotide primers derived frompolynucleotides encoding NAAP are used to amplify DNA using thepolymerase chain reaction (PCR). The DNA may be derived, for example,from diseased or normal tissue, biopsy samples, bodily fluids, and thelike. SNPs in the DNA cause differences in the secondary and tertiarystructures of PCR products in single-stranded form, and thesedifferences are detectable using gel electrophoresis in non-denaturinggels. In fSCCP, the oligonucleotide primers are fluorescently labeled,which allows detection of the amplimers in high-throughput equipmentsuch as DNA sequencing machines. Additionally, sequence databaseanalysis methods, termed in silico SNP (isSNP), are capable ofidentifying polymorphisms by comparing the sequence of individualoverlapping DNA fragments which assemble into a common consensussequence. These computer-based methods filter out sequence variationsdue to laboratory preparation of DNA and sequencing errors usingstatistical models and automated analyses of DNA sequence chromatograms.In the alternative, SNPs may be detected and characterized by massspectrometry using, for example, the high throughput MASSARRAY system(Sequenom, Inc., San Diego Calif.).

SNPs may be used to study the genetic basis of human disease. Forexample, at least 16 common SNPs have been associated withnon-insulin-dependent diabetes mellitus. SNPs are also useful forexamining differences in disease outcomes in monogenic disorders, suchas cystic fibrosis, sickle cell anemia, or chronic granulomatousdisease. For example, variants in the mannose-binding lectin, MBL2, havebeen shown to be correlated with deleterious pulmonary outcomes incystic fibrosis. SNPs also have utility in pharmacogenomics, theidentification of genetic variants that influence a patient's responseto a drug, such as life-threatening toxicity. For example, a variationin N-acetyl transferase is associated with a high incidence ofperipheral neuropathy in response to the anti-tuberculosis drugisoniazid, while a variation in the core promoter of the ALOX5 generesults in diminished clinical response to treatment with an anti-asthmadrug that targets the 5-lipoxygenase pathway. Analysis of thedistribution of SNPs in different populations is useful forinvestigating genetic drift, mutation, recombination, and selection, aswell as for tracing the origins of populations and their migrations(Taylor, J. G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. andZ. Gu (1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr.Opin. Neurobiol. 11:637-641).

Methods which may also be used to quantify the expression of NAAPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves(Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C.et al. (1993) Anal. Biochem. 212:229-236). The speed of quantitation ofmultiple samples maybe accelerated by running the assay in ahigh-throughput format where the oligomer or polynucleotide of interestis presented in various dilutions and a spectrophotometric orcolorimetric response gives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotides described herein may be used as elementson a microarray. The microarray can be used in transcript imagingtechniques which monitor the relative expression levels of large numbersof genes simultaneously as described below. The microarray may also beused to identify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, to monitorprogression/regression of disease as a function of gene expression, andto develop and monitor the activities of therapeutic agents in thetreatment of disease. In particular, this information may be used todevelop a pharmacogenomic profile of a patient in order to select themost appropriate and effective treatment regimen for that patient. Forexample, therapeutic agents which are highly effective and display thefewest side effects may be selected for a patient based on his/herpharmacogenomic profile.

In another embodiment, NAAP, fragments of NAAP, or antibodies specificfor NAAP may be used as elements on a microarray. The microartay may beused to monitor or measure protein-protein interactions, drug-targetinteractions, and gene expression profiles, as described above.

A particular embodiment relates to the use of the polynucleotides of thepresent invention to generate a transcript image of a tissue or celltype. A transcript image represents the global pattern of geneexpression by a particular tissue or cell type. Global gene expressionpatterns are analyzed by quantifing the number of expressed genes andtheir relative abundance under given conditions and at a given time(Seilhamer et al., “Comparative Gene Transcript Analysis,” U.S. Pat. No.5,840,484; hereby expressly incorporated by reference herein). Thus atranscript image may be generated by hybridizing the polynucleotides ofthe present invention or their complements to the totality oftranscripts or reverse transcripts of a particular tissue or cell type.In one embodiment, the hybridization takes place in high-throughputformat, wherein the polynucleotides of the present invention or theircomplements comprise a subset of a plurality of elements on amicroarray. The resultant transcript image would provide a profile ofgene activity.

Transcript images may be generated using transcripts isolated fromtissues, cell lines, biopsies, or other biological samples. Thetranscript image may thus reflect gene expression in vivo, as in thecase of a tissue or biopsy sample, or in vitro, as in the case of a cellline.

Transcript images which profile the expression of the polynucleotides ofthe present invention may also be used in conjunction with in vitromodel systems and preclinical evaluation of pharmaceuticals, as well astoxicological testing of industrial and naturally-occurringenvironmental compounds. All compounds induce characteristic geneexpression patterns, frequently termed molecular fingerprints ortoxicant signatures, which are indicative of mechanisms of action andtoxicity (Nuwaysir, E. F. et al. (1999) Mol. Carcinog. 24:153-159;Steiner, S. and N. L. Anderson (2000) Toxicol. Lett. 112-113:467-471).If a test compound has a signature similar to that of a compound withknown toxicity, it is likely to share those toxic properties. Thesefingerprints or signatures are most useful and refined when they containexpression information from a large number of genes and gene families.Ideally, a genome-wide measurement of expression provides the highestquality signature. Even genes whose expression is not altered by anytested compounds are important as well, as the levels of expression ofthese genes are used to normalize the rest of the expression data. Thenormalization procedure is useful for comparison of expression dataafter treatment with different compounds. While the assignment of genefunction to elements of a toxicant signature aids in interpretation oftoxicity mechanisms, knowledge of gene function is not necessary for thestatistical matching of signatures which leads to prediction of toxicity(see, for example, Press Release 00-02 from the National Institute ofEnvironmental Health Sciences, released Feb. 29, 2000, available athttp://www.niehs.nih.gov/oc/news/toxchip.htm). Therefore, it isimportant and desirable in toxicological screening using toxicantsignatures to include all expressed gene sequences.

In an embodiment, the toxicity of a test compound can be assessed bytreating a biological sample containing nucleic acids with the testcompound. Nucleic acids that are expressed in the treated biologicalsample are hybridized with one or more probes specific to thepolynucleotides of the present invention, so that transcript levelscorresponding to the polynucleotides of the present invention may bequantified. The transcript levels in the treated biological sample arecompared with levels in an untreated biological sample. Differences inthe transcript levels between the two samples are indicative of a toxicresponse caused by the test compound in the treated sample.

Another embodiment relates to the use of the polypeptides disclosedherein to analyze the proteome of a tissue or cell type. The termproteome refers to the global pattern of protein expression in aparticular tissue or cell type. Each protein component of a proteome canbe subjected individually to further analysis. Proteome expressionpatterns, or profiles, are analyzed by quantifying the number ofexpressed proteins and their relative abundance under given conditionsand at a given time. A profile of a cell's proteome may thus begenerated by separating and analyzing the polypeptides of a particulartissue or cell type. In one embodiment, the separation is achieved usingtwo-dimensional gel electrophoresis, in which proteins from a sample areseparated by isoelectric focusing in the first dimension, and thenaccording to molecular weight by sodium dodecyl sulfate slab gelelectrophoresis in the second dimension (Steiner and Anderson, supra).The proteins are visualized in the gel as discrete and uniquelypositioned spots, typically by staining the gel with an agent such asCoomassie Blue or silver or fluorescent stains. The optical density ofeach protein spot is generally proportional to the level of the proteinin the sample. The optical densities of equivalently positioned proteinspots from different samples, for example, from biological sampleseither treated or untreated with a test compound or therapeutic agent,are compared to identify any changes in protein spot density related tothe treatment. The proteins in the spots are partially sequenced using,for example, standard methods employing chemical or enzymatic cleavagefollowed by mass spectrometry. The identity of the protein in a spot maybe determined by comparing its partial sequence, preferably of at least5 contiguous amino acid residues, to the polypeptide sequences ofinterest. In some cases, further sequence data may be obtained fordefinitive protein identification.

A proteomic profile may also be generated using antibodies specific forNAAP to quantify the levels of NAAP expression. In one embodiment, theantibodies are used as elements on a microarray, and protein expressionlevels are quantified by exposing the microarray to the sample anddetecting the levels of protein bound to each array element (Lueking, A.et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L. G. et al. (1999)Biotechniques 27:778-788). Detection maybe performed by a variety ofmethods known in the art, for example, by reacting the proteins in thesample with a thiol- or amino-reactive fluorescent compound anddetecting the amount of fluorescence bound at each array element.

Toxicant signatures at the proteome level are also useful fortoxicological screening, and should be analyzed in parallel withtoxicant signatures at the transcript level. There is a poor correlationbetween transcript and protein abundances for some proteins in sometissues (Anderson, N. L. and J. Seilhamer (1997) Electrophoresis18:533-537), so proteome toxicant signatures may be useful in theanalysis of compounds which do not significantly affect the transcriptimage, but which alter the proteomic profile. In addition, the analysisof transcripts in body fluids is difficult, due to rapid degradation ofmRNA, so proteomic profiling may be more reliable and informative insuch cases.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins that are expressed in the treated biological sample areseparated so that the amount of each protein can be quantified. Theamount of each protein is compared to the amount of the correspondingprotein in an untreated biological sample. A difference in the amount ofprotein between the two samples is indicative of a toxic response to thetest compound in the treated sample. Individual proteins are identifiedby sequencing the amino acid residues of the individual proteins andcomparing these partial sequences to the polypeptides of the presentinvention.

In another embodiment, the toxicity of a test compound is assessed bytreating a biological sample containing proteins with the test compound.Proteins from the biological sample are incubated with antibodiesspecific to the polypeptides of the present invention. The amount ofprotein recognized by the antibodies is quantified. The amount ofprotein in the treated biological sample is compared with the amount inan untreated biological sample. A difference in the amount of proteinbetween the two samples is indicative of a toxic response to the testcompound in the treated sample.

Microarrays may be prepared, used, and analyzed using methods known inthe art (Brennan, T. M. et al. (1995) U.S. Pat. No. 5,474,796; Schena,M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619;Baldeschweiler et al. (1995) PCT application WO95/251116; Shalon, P. etal. (1995) PCT application WO95/35505; Heller, R. A. et al. (1997) Proc.Natl. Acad. Sci. USA 94:2150-2155; Heller, M. J. et al. (1997) U.S. Pat.No. 5,605,662). Various types of microarrays are well known andthoroughly described in Schena, M., ed. (1999; DNA Microarrays: APractical Approach, Oxford University Press, London).

In another embodiment of the invention, nucleic acid sequences encodingNAAP may be used to generate hybridization probes useful in mapping thenaturally occurring genoric sequence. Either coding or noncodingsequences may be used, and in some instances, noncoding sequences may bepreferable over coding sequences. For example, conservation of a codingsequence among members of a multi-gene family may potentially causeundesired cross hybridization during chromosomal mapping. The sequencesmay be mapped to a particular chromosome, to a specific region of achromosome, or to artificial chromosome constructions, e.g., humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions, orsingle chromosome cDNA libraries (Harrington, J. J. et al. (1997) Nat.Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; Trask, B.J. (1991) Trends Genet. 7:149-154). Once mapped, the nucleic acidsequences may be used to develop genetic linkage maps, for example,which correlate the inheritance of a disease state with the inheritanceof a particular chromosome region or restriction fragment lengthpolymorphism (RFLP) (Lander, E. S. and D. Botstein (1986) Proc. Natl.Acad. Sci. USA 83:7353-7357).

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical and genetic map data (Heinz-Ulrich, et al. (1995) in Meyers,supra, pp. 965-968). Examples of genetic map data can be found invarious scientific journals or at the Online Mendelian Inheritance inMan (OMIM) World Wide Web site. Correlation between the location of thegene encoding NAAP on a physical map and a specific disorder, or apredisposition to a specific disorder, may help define the region of DNAassociated with that disorder and thus may further positional cloningefforts.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the exact chromosomal locus is notknown. This information is valuable to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once the gene or genes responsible for a disease or syndromehave been crudely localized by genetic linkage to a particular genomicregion, e.g., ataxia-telangiectasia to 11q22-23, any sequences mappingto that area may represent associated or regulatory genes for furtherinvestigation (Gatti, R. A. et al. (1988) Nature 336:577-580). Thenucleotide sequence of the instant invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, NAAP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between NAAPand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest (Geysen, et al. (1984) PCT application WO84/03564). In thismethod, large numbers of different small test compounds are synthesizedon a solid substrate. The test compounds are reacted with NAAP, orfragments thereof, and washed. Bound NAAP is then detected by methodswell known in the art. Purified NAAP can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NAAP specificallycompete with a test compound for binding NAAP. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NAAP.

In additional embodiments, the nucleotide sequences which encode NAAPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever.

The disclosures of all patents, applications, and publications mentionedabove and below, including U.S. Ser. No. 60/348,442, U.S. Ser. No.60/337,535, U.S. Ser. No. 60/335,544, U.S. Ser. No. 60/344,650, and U.S.Ser. No. 60/334,762, are hereby expressly incorporated by reference.

EXAMPLES

I. Construction of cDNA Libraries

Incyte cDNAs were derived from cDNA libraries described in the LIFESEQGOLD database (Incyte Genomics, Palo Alto Calif.). Some tissues werehomogenized and lysed in guanidinium isothiocyanate, while others werehomogenized and lysed in phenol or in a suitable mixture of denaturants,such as TRIZOL (Invitrogen), a monophasic solution of phenol andguanidine isothiocyanate. The resulting lysates were centrifuged overCsCl cushions or extracted with chloroform. RNA was precipitated fromthe lysates with either isopropanol or sodium acetate and ethanol, or byother routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A)+ RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN,Chatsworth Calif.), or an OLIGOTEX mRNA purification kit (QIAGEN).Alternatively, RNA was isolated directly from tissue lysates using otherRNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion,Austin Tex.).

In some cases, Stratagene was provided with RNA and constructed thecorresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNAlibraries were constructed with the UNIZAP vector system (Stratagene) orSUPERSCRWET plasmid system (Invitrogen), using the recommendedprocedures or similar methods known in the art (Ausubel et al., supra,ch. 5). Reverse transcription was initiated using oligo d(T) or randomprimers. Synthetic oligonucleotide adapters were ligated to doublestranded cDNA, and the cDNA was digested with the appropriaterestriction enzyme or enzymes. For most libraries, the cDNA wassize-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B, orSEPHAROSE CL4B column chromatography (Amersham Biosciences) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of a suitableplasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid(Invitrogen, Carlsbad Calif.), PCDNA2.1 plasmid (Invitrogen), PBK-CMVplasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICISplasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto Calif.), pRARE(Incyte Genomics), or pINCY (Incyte Genomics), or derivatives thereof.Recombinant plasmids were transformed into competent E. coli cellsincluding XL1-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5α, DH10B,or ElectroMAX DH10B from Invitrogen.

II. Isolation of cDNA Clones

Plasmids obtained as described in Example I were recovered from hostcells by in vivo excision using the UNIZAP vector system (Stratagene) orby cell lysis. Plasmids were purified using at least one of thefollowing: a Magic or WIZARD Minipreps DNA purification system(Promega); an AGTC Miniprep purification kit (Edge Biosystems,Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96plasmid purification kit from QIAGEN. Following precipitation, plasmidswere resuspended in 0.1 ml of distilled water and stored, with orwithout lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format (Rao, V. B. (1994) Anal.Biochem. 216:1-14). Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN dye (MolecularProbes, Eugene Oreg.) and a FLUOROSKAN II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

Incyte cDNA recovered in plasmids as described in Example 11 weresequenced as follows. Sequencing reactions were processed using standardmethods or high-throughput instrumentation such as the ABI CATALYST 800(Applied Biosystems) thermal cycler or the PTC-200 thermal cycler (MJResearch) in conjunction with the HYDRA microdispenser (RobbinsScientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNAsequencing reactions were prepared using reagents provided by AmershamBiosciences or supplied in ABI sequencing kits such as the ABI PRISMBIGDYE Terminator cycle sequencing ready reaction kit (AppliedBiosystems). Electrophoretic separation of cDNA sequencing reactions anddetection of labeled polynucleotides were carried out using the MEGABACE1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or377 sequencing system (Applied Biosystems) in conjunction with standardABI protocols and base calling software; or other sequence analysissystems known in the art. Reading frames within the cDNA sequences wereidentified using standard methods (Ausubel et al., supra, ch. 7). Someof the cDNA sequences were selected for extension using the techniquesdisclosed in Example VIII.

The polynucleotide sequences derived from Incyte cDNAs were validated byremoving vector, linker, and poly(A) sequences and by masking ambiguousbases, using algorithms and programs based on BLAST, dynamicprogramming, and dinucleotide nearest neighbor analysis. The Incyte cDNAsequences or translations thereof were then queried against a selectionof public databases such as the GenBank primate, rodent, mammalian,vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM;PROTEOME databases with sequences from Homo sapiens, Rattus norvegicus,Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae,Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, PaloAlto Calif.); hidden Markov model (HMM)-based protein family databasessuch as PFAM, INCY, and TIGRFAM (Haft, D. H. et al. (2001) Nucleic AcidsRes. 29:41-43); and HMM-based protein domain databases such as SMART(Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864;Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244). (HMM is aprobabilistic approach which analyzes consensus primary structures ofgene families; see, for example, Eddy, S. R. (1996) Curr. Opin. Struct.Biol. 6:361-365.) The queries were performed using programs based onBLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences wereassembled to produce full length polynucleotide sequences.Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,stretched sequences, or Genscan-predicted coding sequences (see ExamplesIV and V) were used to extend Incyte cDNA assemblages to full length.Assembly was performed using programs based on Phred, Phrap, and Consed,and cDNA assemblages were screened for open reading frames usingprograms based on GeneMark, BLAST, and FASTA. The full lengthpolynucleotide sequences were translated to derive the correspondingfull length polypeptide sequences. Alternatively, a polypeptide maybegin at any of the methionine residues of the full length translatedpolypeptide. Full length polypeptide sequences were subsequentlyanalyzed by querying against databases such as the GenBank proteindatabases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS,DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein familydatabases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domaindatabases such as SMART. Full length polynucleotide sequences are alsoanalyzed using MACDNASIS PRO software (MiraiBio, Alameda Calif.) andLASERGENE software (DNASTAR). Polynucleotide and polypeptide sequencealignments are generated using default parameters specified by theCLUSTAL algorithm as incorporated into the MEGALIGN multisequencealignment program (DNASTAR), which also calculates the percent identitybetween aligned sequences.

Table 7 summarizes the tools, programs, and algorithms used for theanalysis and assembly of Incyte cDNA and full length sequences andprovides applicable descriptions, references, and threshold parameters.The first column of Table 7 shows the tools, programs, and algorithmsused, the second column provides brief descriptions thereof, the thirdcolumn presents appropriate references, all of which are incorporated byreference herein in their entirety, and the fourth column presents,where applicable, the scores, probability values, and other parametersused to evaluate the strength of a match between two sequences (thehigher the score or the lower the probability value, the greater theidentity between two sequences).

The programs described above for the assembly and analysis of fulllength polynucleotide and polypeptide sequences were also used toidentify polynucleotide sequence fragments from SEQ ID NO:59-116.Fragments from about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies are described in Table 4,column 2.

IV. Identification and Editing of Coding Sequences from Genornic DNA

Putative nucleic acid-associated proteins were initially identified byrunning the Genscan gene identification program against public genomicsequence databases (e.g., gbpri and gbhtg). Genscan is a general-purposegene identification program which analyzes genomic DNA sequences from avariety of organisms (Burge, C. and S. Karlin (1997) J. Mol. Biol.268:78-94; Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol.8:346-354). The program concatenates predicted exons to form anassembled cDNA sequence extending from a methionine to a stop codon. Theoutput of Genscan is a FASTA database of polynucleotide and polypeptidesequences. The maximum range of sequence for Genscan to analyze at oncewas set to 30 kb. To determine which of these Genscan predicted cDNAsequences encode nucleic acid-associated proteins, the encodedpolypeptides were analyzed by querying against PFAM models for nucleicacid-associated proteins. Potential nucleic acid-associated proteinswere also identified by homology to Incyte cDNA sequences that had beenannotated as nucleic acid-associated proteins. These selectedGenscan-predicted sequences were then compared by BLAST analysis to thegenpept and gbpri public databases. Where necessary, theGenscan-predicted sequences were then edited by comparison to the topBLAST hit from genpept to correct errors in the sequence predicted byGenscan, such as extra or omitted exons. BLAST analysis was also used tofind any Incyte cDNA or public cDNA coverage of the Genscan-predictedsequences, thus providing evidence for transcription. When Incyte cDNAcoverage was available, this information was used to correct or confirmthe Genscan predicted sequence. Full length polynucleotide sequenceswere obtained by assembling Genscan-predicted coding sequences withIncyte cDNA sequences and/or public cDNA sequences using the assemblyprocess described in Example III. Alternatively, full lengthpolynucleotide sequences were derived entirely from edited or uneditedGenscan-predicted coding sequences.

V. Assembly of Genomic Sequence Data with cDNA Sequence Data “Stitched”Sequences

Partial cDNA sequences were extended with exons predicted by the Genscangene identification program described in Example IV. Partial cDNAsassembled as described in Example m were mapped to genomic DNA andparsed into clusters containing related cDNAs and Genscan exonpredictions from one or more genomic sequences. Each cluster wasanalyzed using an algorithm based on graph theory and dynamicprogramming to integrate cDNA and genomic information, generatingpossible splice variants that were subsequently confirmed, edited, orextended to create a full length sequence. Sequence intervals in whichthe entire length of the interval was present on more than one sequencein the cluster were identified, and intervals thus identified wereconsidered to be equivalent by transitivity. For example, if an intervalwas present on a cDNA and two genomic sequences, then all threeintervals were considered to be equivalent. This process allowsunrelated but consecutive genomic sequences to be brought together,bridged by cDNA sequence. Intervals thus identified were then “stitched”together by the stitching algorithm in the order that they appear alongtheir parent sequences to generate the longest possible sequence, aswell as sequence variants. Linkages between intervals which proceedalong one type of parent sequence (cDNA to cDNA or genomic sequence togenomic sequence) were given preference over linkages which changeparent type (cDNA to genomic sequence). The resultant stitched sequenceswere translated and compared by BLAST analysis to the genpept and gbpripublic databases. Incorrect ekons predicted by Genscan were corrected bycomparison to the top BLAST hit from genpept. Sequences were furtherextended with additional cDNA sequences, or by inspection of genomicDNA, when necessary.

“Stretched” Sequences

Partial DNA sequences were extended to full length with an algorithmbased on BLAST analysis. First, partial cDNAs assembled as described inExample III were queried against public databases such as the GenBankprimate, rodent, mammalian, vertebrate, and eukaryote databases usingthe BLAST program. The nearest GenBank protein homolog was then comparedby BLAST analysis to either Incyte cDNA sequences or GenScan exonpredicted sequences described in Example IV. A chimeric protein wasgenerated by using the resultant high-scoring segment pairs (HSPs) tomap the translated sequences onto the GenBank protein homolog.Insertions or deletions may occur in the chimeric protein with respectto the original GenBank protein homolog. The GenBank protein homolog,the chimeric protein, or both were used as probes to search forhomologous genomic sequences from the public human genome databases.Partial DNA sequences were therefore “stretched” or extended by theaddition of homologous genomic sequences. The resultant stretchedsequences were examined to determine whether it contained a completegene.

VI. Chromosomal Mapping of NAAP Encoding Polynucleotides

The sequences which were used to assemble SEQ ]ID NO:59-116 werecompared with sequences from the Incyte LIFESEQ database and publicdomain databases using BLAST and other implementations of theSmith-Waterman algorithm. Sequences from these databases that matchedSEQ ID NO:59-116 were assembled into clusters of contiguous andoverlapping sequences using assembly algorithms such as Phrap (Table 7).Radiation hybrid and genetic mapping data available from publicresources such as the Stanford Human Genome Center (SHGC), WhiteheadInstitute for Genome Research (WIGR), and Genethon were used todetermine if any of the clustered sequences had been previously mapped.Inclusion of a mapped sequence in a cluster resulted in the assignmentof all sequences of that cluster, including its particular SEQ ID NO:,to that map location.

Map locations are represented by ranges, or intervals, of humanchromosomes. The map position of an interval, in centiMorgans, ismeasured relative to the terminus of the chromosome's p-arm. (Thecentimorgan (cM) is a unit of measurement based on recombinationfrequencies between chromosomal markers. On average, 1 cM is roughlyequivalent to 1 megabase (Mb) of DNA in humans, although this can varywidely due to hot and cold spots of recombination.) The cM distances arebased on genetic markers mapped by Genethon which provide boundaries forradiation hybrid markers whose sequences were included in each of theclusters. Human genome maps and other resources available to the public,such as the NCBI “GeneMap'99” World Wide Web site(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine ifpreviously identified disease genes map within or in proximity to theintervals indicated above.

VII. Analysis of Polynucleotide Expression

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook and Russell, supra, ch. 7;Ausubel et al., supra, ch. 4).

Analogous computer techniques applying BLAST were used to search foridentical or related molecules in databases such as GenBank or LEESEQ(Incyte Genomics). This analysis is much faster than multiplemembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or similar. The basis of the search is theproduct score, which is defined as:$\frac{{BLAST}\quad{Score} \times {Percent}\quad{Identity}}{5 \times {minimum}\quad\left\{ {{{length}\left( {{Seq}.\quad 1} \right)},{{length}\left( {{Seq}.\quad 2} \right)}} \right\}}$The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. The productscore is a normalized value between 0 and 100, and is calculated asfollows: the BLAST score is multiplied by the percent nucleotideidentity and the product is divided by (5 times the length of theshorter of the two sequences). The BLAST score is calculated byassigning a score of +5 for every base that matches in a high-scoringsegment pair (HSP), and −4 for every mismatch. Two sequences may sharemore than one HSP (separated by gaps). If there is more than one HSP,then the pair with the highest BLAST score is used to calculate theproduct score. The product score represents a balance between fractionaloverlap and quality in a BLAST alignment. For example, a product scoreof 100 is produced only for 100% identity over the entire length of theshorter of the two sequences being compared. A product score of 70 isproduced either by 100% identity and 70% overlap at one end, or by 88%identity and 100% overlap at the other. A product score of 50 isproduced either by 100% identity and 50% overlap at one end, or 79%identity and 100% overlap.

Alternatively, polynucleotides encoding NAAP are analyzed with respectto the tissue sources from which they were derived. For example, somefull length sequences are assembled, at least in part, with overlappingIncyte cDNA sequences (see Example III). Each cDNA sequence is derivedfrom a cDNA library constructed from a human tissue. Each human tissueis classified into one of the following organ/tissue categories:cardiovascular system; connective tissue; digestive system; embryonicstructures; endocrine system; exocrine glands; genitalia, female;genitalia, male; germ cells; hemic and immune system; liver;musculoskeletal system; nervous system; pancreas; respiratory system;sense organs; skin; stomatognathic system; unclassified/mixed; orurinary tract. The number of libraries in each category is counted anddivided by the total number of libraries across all categories.Similarly, each human tissue is classified into one of the followingdisease/condition categories: cancer, cell line, developmental,inflammation, neurological, trauma, cardiovascular, pooled, and other,and the number of libraries in each category is counted and divided bythe total number of libraries across all categories. The resultingpercentages reflect the tissue- and disease-specific expression of cDNAencoding NAAP. cDNA sequences and cDNA library/tissue information arefound in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.).

VIII. Extension of NAAP Encoding Polynucleotides

Full length polynucleotides are produced by extension of an appropriatefragment of the full length molecule using oligonucleotide primersdesigned from this fragment. One primer was synthesized to initiate 5′extension of the known fragment, and the other primer was synthesized toinitiate 3′ extension of the known fragment. The initial primers weredesigned using OLIGO 4.06 software (National Biosciences), or anotherappropriate program, to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

Selected human cDNA libraries were used to extend the sequence. If morethan one extension was necessary or desired, additional or nested setsof primers were designed.

High fidelity amplification was obtained by PCR using methods well knownin the art. PCR was performed in 96-well plates using the PTC-200thermal cycler (MJ Research, Inc.). The reaction mix contained DNAtemplate, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and 2-mercaptoethanol, Taq DNA polymerase (AmershamBiosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68° C., 5 min; Step 7: storage at 4° C. In the alternative, theparameters for primer pair T7 and SK+ were as follows: Step 1: 94° C., 3min; Step 2: 94° C., 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C., 2min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68° C., 5 min;Step 7: storage at 40C.

The concentration of DNA in each well was determined by dispensing 100μl PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; MolecularProbes, Eugene Oreg.) dissolved in 1× TE and 0.5 μl of undiluted PCRproduct into each well of an opaque fluorimeter plate (Corning Costar,Acton Mass.), allowing the DNA to bind to the reagent. The plate wasscanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measurethe fluorescence of the sample and to quantify the concentration of DNA.A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a 1% agarose gel to determine which reactions weresuccessful in extending the sequence.

The extended nucleotides were desalted and concentrated, transferred to384-well plates, digested with CviJI cholera virus endonuclease(Molecular Biology Research, Madison Wis.), and sonicated or shearedprior to religation into pUC 18 vector (Amershamn Biosciences). Forshotgun sequencing, the digested nucleotides were separated on lowconcentration (0.6 to 0.8%) agarose gels, fragments were excised, andagar digested with Agar ACE (Promega). Extended clones were religatedusing T4 ligase (New England Biolabs, Beverly Mass.) into pUC 18 vector(Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) tofill-in restriction site overhangs, and transfected into competent E.coli cells. Transformed cells were selected on antibiotic-containingmedia, and individual colonies were picked and cultured overnight at 37°C. in 384-well plates in LB/2× carb liquid media.

The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene)with the following parameters: Step 1: 94° C., 3 min; Step 2: 94° C., 15sec; Step 3: 60 OC, 1 min; Step 4: 72° C., 2 min; Step 5: steps 2, 3,and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7: storage at 4° C.DNA was quantified by PICOGREEN reagent (Molecular Probes) as describedabove. Samples with low DNA recoveries were reamplified using the sameconditions as described above. Samples were diluted with 20%dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energytransfer sequencing primers and the DYENAMIC DIRECT kit (AmershamBiosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Applied Biosystems).

In like manner, full length polynucleotides are verified using the aboveprocedure or are used to obtain 5′ regulatory sequences using the aboveprocedure along with oligonucleotides designed for such extension, andan appropriate genomic library.

IX. Identification of Single Nucleotide Polymorphisms in NAAP EncodingPolynucleotides

Common DNA sequence variants known as single nucleotide polymorphisms(SNPs) were identified in SEQ ID NO:59-116 using the LIFESEQ database(Incyte Genomics). Sequences from the same gene were clustered togetherand assembled as described in Example III, allowing the identificationof all sequence variants in the gene. An algorithm consisting of aseries of filters was used to distinguish SNPs from other sequencevariants. Preliminary filters removed the majority of basecall errors byrequiring a minimum Phred quality score of 15, and removed sequencealignment errors and errors resulting from improper trimming of vectorsequences, chimeras, and splice variants. An automated procedure ofadvanced chromosome analysis analysed the original chromatogram files inthe vicinity of the putative SNP. Clone error filters used statisticallygenerated algorithms to identify errors introduced during laboratoryprocessing, such as those caused by reverse transcriptase, polymerase,or somatic mutation. Clustering error filters used statisticallygenerated algorithms to identify errors resulting from clustering ofclose homologs or pseudogenes, or due to contamination by non-humansequences. A final set of filters removed duplicates and SNPs found inimmunoglobulins or T-cell receptors.

Certain SNPs were selected for further characterization by massspectrometry using the high throughput MASSARRAY system (Sequenom, Inc.)to analyze allele frequencies at the SNP sites in four different humanpopulations. The Caucasian population comprised 92 individuals (46 male,46 female), including 83 from Utah, four French, three Venezualan, andtwo Amish individuals. The African population comprised 194 individuals(97 male, 97 female), all African Americans. The Hispanic populationcomprised 324 individuals (162 male, 162 female), all Mexican Hispanic.The Asian population comprised 126 individuals (64 male, 62 female) witha reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean,5% Vietnamese, and 8% otlier Asian. Allele frequencies were firstanalyzed in the Caucasian population; in some cases those SNPs whichshowed no allelic variance in this population were not further tested inthe other three populations.

X. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:59-116 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham Biosciences), and T4 polynucleotide kinase DuPontNEN, Boston Mass.). The labeled oligonucleotides are substantiallypurified using a SEPHADEX G-25 superfine size exclusion dextran beadcolumn (Amersham Biosciences). An aliquot containing 10⁷ counts perminute of the labeled probe is used in a typical membrane-basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II(DuPont NEN).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham N. H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under conditions of up to, for example, 0.1× saline sodiumcitrate and 0.5% sodium dodecyl sulfate. Hybridization patterns arevisualized using autoradiography or an alternative imaging means andcompared.

XI. Microarrays

The linkage or synthesis of array elements upon a microarray can beachieved utilizing photolithography, piezoelectric printing (ink-jetprinting; see, e.g., Baldeschweiler et al., supra), mechanicalmicrospotting technologies, and derivatives thereof. The substrate ineach of the aforementioned technologies should be uniform and solid witha non-porous surface (Schena, M., ed. (1999) DNA Microarrays: APractical Approach, Oxford University Press, London). Suggestedsubstrates include silicon, silica, glass slides, glass chips, andsilicon wafers. Alternatively, a procedure analogous to a dot or slotblot may also be used to arrange and link elements to the surface of asubstrate using thermal, UV, chemical, or mechanical bonding procedures.A typical array may be produced using available methods and machineswell known to those of ordinary skill in the art and may contain anyappropriate number of elements (Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; Marshall,A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).

Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments oroligomers thereof may comprise the elements of the microarray. Fragmentsor oligomers suitable for hybridization can be selected using softwarewell known in the art such as LASERGENE software (DNASTAR). The arrayelements are hybridized with polynucleotides in a biological sample. Thepolynucleotides in the biological sample are conjugated to a fluorescentlabel or other molecular tag for ease of detection. After hybridization,nonhybridized nucleotides from the biological sample are removed, and afluorescence scanner is used to detect hybridization at each arrayelement. Alternatively, laser desorbtion and mass spectrometry may beused for detection of hybridization. The degree of complementarity andthe relative abundance of each polynucleotide which hybridizes to anelement on the microarray may be assessed. In one embodiment, microarraypreparation and usage is described in detail below.

Tissue or Cell Sample Preparation

Total RNA is isolated from tissue samples using the guanidiniumthiocyanate method and poly(A)⁺ RNA is purified using the oligo-(dT)cellulose method. Each poly(A)⁺ RNA sample is reverse transcribed usingMMLV reverse-transcriptase, 0.05 pg/μl oligo-(dT) primer (21mer), 1×first strand buffer, 0.03 units/μl RNase inhibitor, 500 μM dATP, 500 μMdGTP, 500 μM dTTP, 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5(Amersham Biosciences). The reverse transcription reaction is performedin a 25 ml volume containing 200 ng poly(A)⁺ RNA with GEMBRIGHT kits(Incyte Genomics). Specific control poly(A)⁺ RNAs are synthesized by invitro transcription from non-coding yeast genomic DNA. After incubationat 37° C. for 2 hr, each reaction sample (one with Cy3 and another withCy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide andincubated for 20 minutes at 85° C. to the stop the reaction and degradethe RNA. Samples are purified using two successive CHROMA SPIN 30 gelfiltration spin columns (Clontech, Palo Alto Calif.) and aftercombining, both reaction samples are ethanol precipitated using 1 ml ofglycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol.The sample is then dried to completion using a SpeedVAC (SavantInstruments Inc., Holbrook N.Y.) and resuspended in 14 μl 5×SSC/0.2%SDS.

Microarray Preparation

Sequences of the present invention are used to generate array elements.Each array element is amplified from bacterial cells containing vectorswith cloned cDNA inserts. PCR amplification uses primers complementaryto the vector sequences flanking the cDNA insert. Array elements areamplified in thirty cycles of PCR from an initial quantity of 1-2 ng toa final quantity greater than 5 μg. Amplified array elements are thenpurified using SEPHACRYL-400 (Amersham Biosciences).

Purified array elements are immobilized on polymer-coated glass slides.Glass microscope slides (Corning) are cleaned by ultrasound in 0.1% SDSand acetone, with extensive distilled water washes between and aftertreatments. Glass slides are etched in 4% hydrofluoric acid (VWRScientific Products Corporation (VWR), West Chester Pa.), washedextensively in distilled water, and coated with 0.05% aminopropyl silane(Sigma) in 95% ethanol. Coated slides are cured in a 110° C. oven.

Array elements are applied to the coated glass substrate using aprocedure described in U.S. Pat. No. 5,807,522, incorporated herein byreference. 1 μl of the array element DNA, at an average concentration of100 ng/μl, is loaded into the open capillary printing element by ahigh-speed robotic apparatus. The apparatus then deposits about 5 nl ofarray element sample per slide.

Micro arrays are UV-crosslinked using a STRATALINKER UV-crosslinker(Stratagene). Microarrays are washed at room temperature once in 0.2%SDS and three times in distilled water. Non-specific binding sites areblocked by incubation of microarrays in 0.2% casein in phosphatebuffered saline (PBS) (Tropix, Inc., Bedford Mass.) for 30 minutes at60° C. followed by washes in 0.2% SDS and distilled water as before.

Hybridization

Hybridization reactions contain 9 μl of sample mixture consisting of 0.2μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2%SDS hybridization buffer. The sample mixture is heated to 65° C. for 5minutes and is aliquoted onto the microarray surface and covered with an1.8 cm² coverslip. The arrays are transferred to a waterproof chamberhaving a cavity just slightly larger than a microscope slide. Thechamber is kept at 100% humidity internally by the addition of 140 μl of5×SSC in a corner of the chamber. The chamber containing the arrays isincubated for about 6.5 hours at 60° C. The arrays are washed for 10 minat 45° C. in a first wash buffer (1×SSC, 0.1% SDS), three times for 10minutes each at 45° C. in a second wash buffer (0.1×SSC), and dried.

Detection

Reporter-labeled hybridization complexes are detected with a microscopeequipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., SantaClara Calif.) capable of generating spectral lines at 488 nm forexcitation of Cy3 and at 632 nm for excitation of Cy5. The excitationlaser light is focused on the array using a 20× microscope objective(Nikon, Inc., Melville N.Y.). The slide containing the array is placedon a computer-controlled X-Y stage on the microscope and raster-scannedpast the objective. The 1.8 cm×1.8 cm array used in the present exampleis scanned with a resolution of 20 micrometers.

In two separate scans, a mixed gas multiline laser excites the twofluorophores sequentially. Emitted light is split, based on wavelength,into two photomultiplier tube detectors (PMT R1477, Hamamatsu PhotonicsSystems, Bridgewater N.J.) corresponding to the two fluorophores.Appropriate filters positioned between the array and the photomultipliertubes are used to filter the signals. The emission maxima of thefluorophores used are 565 nm for Cy3 and 650 nm for Cy5. Each array istypically scanned twice, one scan per fluorophore using the appropriatefilters at the laser source, although the apparatus is capable ofrecording the spectra from both fluorophores simultaneously.

The sensitivity of the scans is typically calibrated using the signalintensity generated by a cDNA control species added to the samplemixture at a known concentration. A specific location on the arraycontains a complementary DNA sequence, allowing the intensity of thesignal at that location to be correlated with a weight ratio ofhybridizing species of 1:100,000. When two samples from differentsources (e.g., representing test and control cells), each labeled with adifferent fluorophore, are hybridized to a single array for the purposeof identifying genes that are differentially expressed, the calibrationis done by labeling samples of the calibrating cDNA with the twofluorophores and adding identical amounts of each to the hybridizationmixture.

The output of the photomultiplier tube is digitized using a 12-bitRTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc.,Norwood Mass.) installed in an IBM-compatible PC computer. The digitizeddata are displayed as an image where the signal intensity is mappedusing a linear 20-color transformation to a pseudocolor scale rangingfrom blue (low signal) to red (high signal). The data is also analyzedquantitatively. Where two different fluorophores are excited andmeasured simultaneously, the data are first corrected for opticalcrosstalk (due to overlapping emission spectra) between the fluorophoresusing each fluorophore's emission spectrum.

A grid is superimposed over the fluorescence signal image such that thesignal from each spot is centered in each element of the grid. Thefluorescence signal within each element is then integrated to obtain anumerical value corresponding to the average intensity of the signal.The software used for signal analysis is the GEMTOOLS gene expressionanalysis program (Incyte Genomics). Array elements that exhibit at leastabout a two-fold change in expression, a signal-to-background ratio ofat least about 2.5, and an element spot size of at least about 40%, areconsidered to be differentially expressed.

Expression

SEQ ID NO:65 showed differential expression in cancer cell lines versusnon-cancerous cell lines, as determined by microarray analysis. Forexample, the expression of SEQ ID NO:65 was decreased by at least twofold in breast tumor cell lines each isolated from pleural effusion fromdonors at different stages of tumor progression and malignanttransformation when grown in one of two different chemically defined,serum-free media both supplemented with growth factors and growthhormones. Therefore, SEQ ID NO:65 is useful in diagnostic assays forbreast cancer.

Normal breast cell lines are obtained as follows. Primary mammary glandcells are isolated from a donor with fibrocystic breast disease.Humorous breast cell lines are obtained as follows. Breast carcinomacells are derived in vitro from cells emigrating from a tumor.Alternately, breast tumor cells are isolated from invasive tumor ofdonors. Further, nonmalignant or malignant primary breast adenocarcinomacells are obtained from the pleural effusion of donors.

Further, the expression of SEQ ID NO:65 was decreased at least two-foldin treated human adipocytes from obese and normal donors when comparedto non-treated adipocytes from the same donors. The normal human primarysubcutaneous preadipocytes were isolated from adipose tissue of a28-year-old healthy female with a body mass index (BMI) of 23.59. Theobese human primary subcutaneous preadipocytes were isolated fromadipose tissue of a 40-year-old healthy female with a body mass index(BMI) of 32.47. The preadipocytes were cultured and induced todifferentiate into adipocytes by culturing them in the differentiationmedium containing the active components, PPAR-γ agonist and humaninsulin. Human preadipocytes were treated with human insulin and PPAR-γagonist for three days and subsequently were switched to mediumcontaining insulin for 24 hours, 48 hours, four days, 8 days or 15 daysbefore the cells were collected for analysis. Differentiated adipocyteswere compared to untreated preadipocytes maintained in culture in theabsence of inducing agents. Between 80% and 90% of the preadipocytesfinally differentiated to.adipocytes as observed under phase contrastmicroscope. Thus, SEQ ID NO:65 is useful for the diagnosis, prognosis,or treatment of diabetes mellitus and other disorders, such as obesity,hypertension, atherosclerosis, polycystic ovarian syndrome, and cancersincluding breast, prostate, and colon.

For example, SEQ ID NO:72-74 showed differential expression in tumoroustissue versus non-tumorous tissues, as determined by microarrayanalysis. The expression of cDNAs from lung tumor tissue from severaldonors was compared with that of normal lung tissue from the same donor,respectively. Array elements that exhibited about at least a two-foldchange in expression and a signal intensity over 250 units, asignal-to-background ratio of a least 2.5, and an element spot size ofat least 40% were identified as differentially expressed using theGEMTOOLS program (Incyte Genomics).

The expression of SEQ ID NO:72 was increased at least two-fold in lungsquamous cell carcinoma when matched with normal tissue from the samedonor. The tumorous lung tissue was obtained from the lung of a66-year-old male with lung squamous cell carcinoma. Normal tissue wasobtained from grossly uninvolved lung tissue from the same donor.Therefore, SEQ ID NO:72 is useful in diagnostic assays for lung squamouscell carcinoma.

Alternately, the expression of SEQ ID NO:73 was decreased at least2.7-fold in lung adenocarcinoma when matched with normal tissue from thesame donor. The tumorous lung tissue was obtained from the right lung ofa 60-year old donor with moderately differentiated adenocarcinoma.Normal tissue was obtained from grossly uninvolved tissue from the rightlung from the same donor. Therefore, SEQ ID NO:73 is useful indiagnostic assays for lung adenocarcinoma. Further, the expression ofSEQ ID NO:74 was increased at least 2.7-fold in lung adenocarcinoma whenmatched with normal tissue from the same donor. The tumorous lung tissuewas obtained from the lung of a 66-year old female with lungadenocarcinoma. Normal tissue was obtained from grossly uninvolvedtissue from grossly uninvolved lung tissue from the same donor. Theexpression of SEQ ID NO:74 was increased at least 3.2-fold in lungsquamous cell carcinoma from two donors when matched with normal tissuefrom the same donor. In one case, the tumorous lung tissue was obtainedfrom the lung of a 66-year-old male with lung squamous cell carcinoma.In the other case, the tumorous lung tissue was obtained from the lungof a 73-year old male with lung squamous cell carcinoma. Normal tissuewas obtained from grossly uninvolved lung tissue from the same donor,respectively. Therefore, SEQ ID NO:74 is useful in diagnostic assays forlung adenocarcinoma and squamous cell carcinoma.

For example, SEQ ID NO:79 showed increased expression in colon tissueaffected by colon cancer versus normal colon tissue as determined bymicroarray analysis. Gene expression profiles were obtained by comparingnormal colon tissue from a 67 year-old donor with moderatelydifferentiated adenocarcinoma (Dukes B, TNM classification) tocancer-affected colon tissue from the same donor. Samples were providedby the Huntsman Cancer Institute. Therefore, SEQ ID NO:79 is useful indiagnostic assays for disorders of cell proliferation including coloncancer.

For example, SEQ ID NO:79 showed decreased expression in ovary tissueaffected by ovarian cancer versus normal ovary tissue as determined bymicroarray analysis. A normal ovary from a 79 year-old female donor wascompared to an ovarian tumor from the same donor. Samples were providedby the Huntsman Cancer Institute. Therefore, SEQ ID NO:79 is useful indiagnostic assays for disorders of cell proliferation including ovariancancer.

For example, SEQ ID NO:79 showed decreased expression in C3A cellstreated with dexamethasone, versus untreated C3A cells, as determined bymicroarray analysis. The human C3A cell line is a clonal derivative ofHepG2/C3 (hepatoma cell line, isolated from a 15-year-old male withliver tumor), which was selected for strong contact inhibition ofgrowth. Early Confluent C3A cells were treated with dexamethasone at 1,10, and 100 μM for 1, 3, and 6 hours. The treated cells were compared tountreated early confluent C3A cells. Therefore, SEQ ID NO:79 is usefulin diagnostic assays for, and monitoring treatment of,autoimmune/inflammatory disorders.

For example, SEQ ID NO:81 showed differential expression in fibroblastsaffected by Tangiers Disease (TD) versus normal fibroblasts, when bothwere treated with LDL cholesterol, as determined by microarray analysis.Normal and TD-derived fibroblasts were compared cultured in the presenceof cholesterol and compared with the same cell type cultured in theabsence of cholesterol. Human fibroblasts were obtained from skinexplants from both normal subjects and two patients with homozygous ID.Cell lines were immortalized by transfection with human papillomavirus16 genes E6 and E7 and a neomycin resistance selectable marker, and TDwas confirmed in TD-derived cells by reduced apoA-I mediated tritiatedcholesterol efflux. Therefore, SEQ ID NO:81 is useful in diagnosticassays for autoimmune/inflammatory disorders including Tangier Disease.

For example, SEQ ID NO:93 showed differential expression in mammarycells affected by breast carcinoma versus nonmalignant mammaryepithelial cells as determined by microarray analysis. The geneexpression profile of a nonmalignant mammary epithelial cell line wascompared to the gene expression profiles of breast carcinoma lines atdifferent stages of tumor progression. Cell lines compared included: a)MCF-10A, a breast mammary gland cell line isolated from a 36-year-oldwoman with fibrocystic breast disease; b) MCF7, a nonmalignant breastadenocarcinoma cell line isolated from the pleural effusion of a69-year-old female; c) T-47D, a breast carcinoma cell line isolated froma pleural effusion obtained from a 54-year-old female with aninfiltrating ductal carcinoma of the breast; d) Sk-BR-3, a breastadenocarcinoma cell line isolated from a malignant pleural effusion of a43-year-old female; e) BT-20, a breast carcinoma cell line derived invitro from tumor mass isolated from a 74-year-old female; f) MDA-mb-231,a breast tumor cell line isolated from the pleural effusion of a 51-yearold female; and g) MDA-mb-435S, a spindle shaped strain that evolvedfrom the parent line (435) isolated from the pleural effusion of a 31-year-old female with metastatic, ductal adenocarcinoma of the breast.

The cells were grown in the supplier's recommended medium to 70-80%confluence prior to RNA harvest. Expression was decreased by at leasttwo-fold in 4 of the 6 breast carcinoma cell lines as compared to thenonmalignant mammary epithelial cell line. Therefore, SEQ 1D NO:93 isuseful in diagnostic assays for and monitoring treatment of, cellproliferative disorders including breast carcinoma.

As another example, SEQ ID NO:94 showed decreased expression in tissueaffected by adenocarcinoma versus normal tissue as determined bymicroarray analysis. A sample of tissue right lung tissue that showedmoderately differentiated adenocarcinoma of was compared to grosslyuninvolved lung tissue from the same donor (Huntsman Cancer Institute,Salt Lake City, Utah). Therefore, SEQ ID NO:94 is useful in diagnosticassays for, and monitoring treatment of, cell proliferative disordersincluding adenocarcinoma.

As another example, SEQ ID NO:98 showed decreased expression instimulated dendritic cells treated with CD40 antibodies versusstimulated dendritic cells not treated with CD40 antibodies, asdetermined by microarray analysis. Human monocytic dendritic cells(mDCs) were derived in vitro from the adherent cellular fraction of theperipheral blood of 4 healthy volunteer donors. The adherent leukocytes,mostly monocytes, were incubated for 13 days in the presence ofrecombinant interleukin-4 (10 ng/ml) and granulocyte/macrophage colonystimulating factor (10 ng/ml). The differentiated mDCs were collectedafter 13 days from the non-adherent cellular fraction and activated inthe presence of soluble mouse anti-human CD40 antibodies for 2, 8, and24 hours. The anti-CD40 treated mDCs were compared to untreated mDCs.Therefore, SEQ ED NO:98 is useful in diagnostic assays for, andmonitoring treatment of, autoimmune/inflammatory disorders.

As another example, SEQ ID NO:100 showed decreased expression in cellstreated with tumor necrosis factor alpha TNF-α), which mediates theinflammatory response through activation of signal transductionpathways, versus untreated cells as determined by microarray analysis.Human aortic endothelial cells (HMVECdNeos) were grown to 85% confluenceand then treated for 1, 2, 4, 8, and 24 hours with tumor necrosis factoralpha (TNF-α). TNF-α -treated cells were compared to untreatedHMVECdNeos collected at 85% confluence (0 hour). Therefore, SEQ IDNO:100 is useful in diagnostic assays for, and monitoring treatment of,cell proliferative disorders.

In order to evaluate RNA expression, HMVECdNeo cells were grown to 85%confluency and then treated with TNF-α (10 ng/ml) for 2, 4, 8, and 24hours. TNF-α-treated cells were compared to untreated HMVECdNeoscollected at 85% confluency (0 hour). The expression of SEQ ID NO:108was underexpressed by at least two-fold in TNF-α-treated versusuntreated cells at the last three time points tested. Therefore, SEQ IDNO:108 maybe useful in disease staging and diagnostic assays for cellproliferative and inflammatory disorders, including those involvingnucleic acid-associated proteins.

Region-specific RNA expression in human brain tissue was evaluated usingspecific dissected brain regions from a non-demented human female brain.Brain regions were then pooled and used as the control. Specific brainregions were then compared to the mixed brain control. The mixed braincontrol was reconstituted from the purified mRNA isolated from the majorregions of the brain. The expression of SEQ ID NO:109 was underexpressedby at least two-fold in the dentate nuclear brain tissue as compared tothe mixed brain control tissue. Therefore, SEQ ID NO:109 maybe useful indisease staging and diagnostic assays for cell proliferative and/orneurological disorders, including those involving nucleicacid-associated proteins.

XII. Complementary Polynucleotides

Sequences complementary to the NAAP-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring NAAP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of NAAP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the NAAP-encoding transcript.

XIII. Expression of NAAP

Expression and purification of NAAP is achieved using bacterial orvirus-based expression systems. For expression of NAAP in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express NAAP uponinduction with isopropyl beta-D-thiogalactopyranoside (WIUG). Expressionof NAAP in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding NAAP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus (Engelhard, E. K. et al. (1994)Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.Gene Ther. 7:1937-1945).

In most expression systems, NAAP is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (AmershamBiosciences). Following purification, the GST moiety can beproteolytically cleaved from NAAP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingconmmercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel et al.(supra, ch. 10 and 16). Purified NAAP obtained by these methods can beused directly in the assays shown in Examples XVII, XVIII, XIX, and XX,where applicable.

XIV. Functional Assays

NAAP function is assessed by expressing the sequences encoding NAAP atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude PCMV SPORT plasmid (Invitrogen, Carlsbad Calif.) and PCR3.1plasmid (Invitrogen), both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, for example, an endothelial or hematopoietic cellline, using either liposome formulations or electroporation. 1-2 μg ofan additional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP and to evaluate the apoptotic state ofthe cells and other cellular properties. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York N.Y.).

The influence of NAAP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding NAAPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using 30 magnetic beads coated with either humanIgG or antibody against CD64 (DYNAL, Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art.

Expression of mRNA encoding NAAP and other genes of interest can beanalyzed by northern analysis or microarray techniques.

XV. Production of NAAP Specific Antibodies

NAAP substantially purified using polyacrylamide gel electrophoresis(PAGE; see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizeanimals (e.g., rabbits, mice, etc.) and to produce antibodies usingstandard protocols.

Alternatively, the NAAP amino acid sequence is analyzed using LASERGENEsoftware (DNASTAR) to determine regions of high immunogenicity, and acorresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art (Ausubel et al.,supra, ch. 11).

Typically, oligopeptides of about 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Applied Biosystems) using FMOCchemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MB S) to increaseimmunogenicity (Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide and anti-NAAP activity by, forexample, binding the peptide or NAAP to a substrate, blocking with 1%BSA, reacting with rabbit antisera, washing, and reacting withradio-iodinated goat anti-rabbit IgG.

XVI. Purification of Naturally Occurring NAAP Using Specific Antibodies

Naturally occurring or recombinant NAAP is substantially purified byimmunoaffinity chromatography using antibodies specific for NAAP. Animmunoaffinity column is constructed by covalently coupling anti-NAAPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Biosciences). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing NAAP are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof NAAP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NAAP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and NAAPis collected.

XVII. Identification of Molecules Which Interact with NAAP

NAAP, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133:529-539). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled NAAP, washed, and anywells with labeled NAAP complex are assayed. Data obtained usingdifferent concentrations of NAAP are used to calculate values for thenumber, affinity, and association of NAAP with the candidate molecules.

Alternatively, molecules interacting with NAAP are analyzed using theyeast two-hybrid system as described in Fields, S. and O. Song (1989;Nature 340:245-246), or using commercially available kits based on thetwo-hybrid system, such as the MATCHMAKER system (Clontech).

NAAP may also be used in the PATHCALLING process (CuraGen Corp., NewHaven Conn.) which employs the yeast two-hybrid system in ahigh-throughput manner to determine all interactions between theproteins encoded by two large libraries of genes (Nandabalan, K. et al.(2000) U.S. Pat. No. 6,057,101).

XVIII. Demonstration of NAAP Activity

NAAP activity is measured by its ability to stimulate transcription of areporter gene (Liu, H. Y. et al. (1997) EMBO J. 16:5289-5298). The assayentails the use of a well characterized reporter gene construct,LexA_(op)-LacZ, that consists of LexA DNA transcriptional controlelements (LexA_(op)) fused to sequences encoding the E. coli LacZenzyme. The methods for constructing and expressing fusion genes,introducing them into cells, and measuring LacZ enzyme activity, arewell known to those skilled in the art. Sequences encoding NAAP arecloned into a plasmid that directs the synthesis of a fusion protein,LexA-NAAP, consisting of NAAP and a DNA binding domain derived from theLexA transcription factor. The resulting plasmid, encoding a LexA-NAAPfusion protein, is introduced into yeast cells along with a plasmidcontaining the LexA_(op)-LacZ reporter gene. The amount of LacZ enzymeactivity associated with LexA-NAAP transfected cells, relative tocontrol cells, is proportional to the amount of transcription stimulatedby the NAAP.

Alternatively, NAAP activity is measured by its ability to bind zinc. A5-10 μM sample solution in 2.5 mM ammonium acetate solution at pH 7.4 iscombined with 0.05 M zinc sulfate solution (Aldrich, Milwaukee Wis.) inthe presence of 100 μM dithiothreitol with 10% methanol added. Thesample and zinc sulfate solutions are allowed to incubate for 20minutes. The reaction solution is passed through a VYDAC column (GraceVydac, Hesperia, Calif.) with approximately 300 Angstrom bore size and 5μM particle size to isolate zinc-sample complex from the solution, andinto a mass spectrometer (PE Sciex, Ontario, Canada). Zinc bound tosample is quantified using the functional atomic mass of 63.5 Daobserved by Whittal, R. M. et al. ((2000) Biochemistry 39:8406-8417).

In the alternative, a method to determine nucleic acid binding activityof NAAP involves a polyacrylamide gel mobility-shift assay. Inpreparation for this assay, NAAP is expressed by transforming amammalian cell line such as COS7, HeLa or CHO with a eukaryoticexpression vector containing NAAP cDNA. The cells are incubated for48-72 hours after transformation under conditions appropriate for thecell line to allow expression and accumulation of NAAP. Extractscontaining solubilized proteins can be prepared from cells expressingNAAP by methods well known in the art. Portions of the extractcontaining NAAP are added to [³²P]-labeled RNA or DNA. Radioactivenucleic acid can be synthesized in vitro by techniques well known in theart. The mixtures are incubated at 25° C. in the presence of RNase- andDNase-inhibitors under buffered conditions for 5-10 minutes. Afterincubation, the samples are analyzed by polyacrylamide gelelectrophoresis followed by autoradiography. The presence of a band onthe autoradiogram indicates the formation of a complex between NAAP andthe radioactive transcript. A band of similar mobility will not bepresent in samples prepared using control extracts prepared fromuntransformed cells.

In the alternative, a method to determine methylase activity of NAAPmeasures transfer of radiolabeled methyl groups between a donorsubstrate and an acceptor substrate. Reaction mixtures (50 μl finalvolume) contain 15 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM dithiothreitol,3% polyvinylalcohol, 1.5 μCi [methyl-³H]AdoMet (0.375 μM AdoMet)(DuPont-NEN), 0.6 μg NAAP, and acceptor substrate (e.g., 0.4 μg[³⁵S]RNA, or 6-mercaptopurine (6-MP) to 1 mM final concentration).Reaction mixtures are incubated at 30° C. for 30 minutes, then 65° C.for 5 minutes.

Analysis of [methyl-³H]RNA is as follows: (1) 50 μl of 2× loading buffer(20 mM Tris-HCl, pH 7.6, 1 M LiCl, 1 mM EDTA, 1% sodium dodecyl sulphate(SDS)) and 50 μl oligo d(T)-cellulose (10 mg/ml in 1× loading buffer)are added to the reaction mixture, and incubated at ambient temperaturewith shaking for 30 minutes. (2) Reaction mixtures are transferred to a96-well filtration plate attached to a vacuum apparatus. (3) Each sampleis washed sequentially with three 2.4 ml aliquots of 1× oligo d(T)loading buffer containing 0.5% SDS, 0.1% SDS, or no SDS. (4) RNA iseluted with 300 μl of water into a 96-well collection plate, transferredto scintillation vials containing liquid scintillant, and radioactivitydetermined.

Analysis of [methyl-³H]6-MP is as follows: (1) 500 μl 0.5 M boratebuffer, pH 10.0, and then 2.5 ml of 20% (v/v) isoamyl alcohol in tolueneare added to the reaction mixtures. (2) The samples are mixed byvigorous vortexing for ten seconds. (3) After centrifugation at 700g for10 minutes, 1.5 ml of the organic phase is transferred to scintillationvials containing 0.5 ml absolute ethanol and liquid scintillant, andradioactivity determined. (4) Results are corrected for the extractionof 6-MP into the organic phase (approximately 41%).

In the alternative, type I topoisomerase activity of NAAP can be assayedbased on the relaxation of a supercoiled DNA substrate. NAAP isincubated with its substrate in a buffer lacking Mg²⁺ and ATP?, thereaction is terminated, and the products are loaded on an agarose gel.Altered topoisomers can be distinguished from supercoiled substrateelectrophoretically. This assay is specific for type I topoisomeraseactivity because Mg²⁺ and ATP are necessary cofactors for type IItopoisomerases.

Type II topoisomerase activity of NAAP can be assayed based on thedecatenation of a kinetoplast DNA (KDNA) substrate. NAAP is incubatedwith KDNA, the reaction is terminated, and the products are loaded on anagarose gel. Monomeric circular KDNA can be distinguished from catenatedKDNA electrophoretically. Kits for measuring type I and type IItopoisomerase activities are available commercially from Topogen(Columbus Ohio).

ATP-dependent RNA helicase unwinding activity of NAAP can be measured bythe method described by Zhang and Grosse (1994; Biochemistry33:3906-3912). The substrate for RNA unwinding consists of ³²P-labeledRNA composed of two RNA strands of 194 and 130 nucleotides in lengthcontaining a duplex region of 17 base-pairs. The RNA substrate isincubated together with ATP, Mg²⁺, and varying amounts of NAAP in aTris-HCl buffer, pH 7.5, at 37° C. for 30 minutes. The single-strandedRNA product is then separated from the double-stranded RNA substrate byelectrophoresis through a 10% SDS-polyacrylamide gel, and quantitated byautoradiography. The amount of single-stranded RNA recovered isproportional to the amount of NAAP in the preparation.

In the alternative, NAAP function is assessed by expressing thesequences encoding NAAP at physiologically elevated levels in mammaliancell culture systems. cDNA is subcloned into a mammalian expressionvector containing a strong promoter that drives high levels of cDNAexpression. Vectors of choice include pCMV SPORT (Life Technologies) andpCR3.1 (Invitrogen Corporation, Carlsbad Calif.), both of which containthe cytomegalovirus promoter. 5-10 μg of recombinant vector aretransiently transfected into a human cell line, preferably ofendothelial or hematopoietic origin, using either liposome formulationsor electroporation. 1-2 μg of an additional plasmid containing sequencesencoding a marker protein are co-transfected.

Expression of a marker protein provides a means to distinguishtransfected cells from nontransfected cells and is a reliable predictorof cDNA expression from the recombinant vector. Marker proteins ofchoice include, e.g., Green Fluorescent Protein (GFP; CLONTECH), CD64,or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP and to evaluate the apoptotic state of the cells andother cellular properties.

FCM detects and quantifies the uptake of fluorescent molecules thatdiagnose events preceding or coincident with cell death. These eventsinclude changes in nuclear DNA content as measured by staining of DNAwith propidium iodide; changes in cell size and granularity as measuredby forward light scatter and 90 degree side light scatter;down-regulation of DNA synthesis as measured by decrease inbromodeoxyuridine uptake; alterations in expression of cell surface andintracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York N.Y.

The influence of NAAP on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding NAAPand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Inc., Lake Success N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding NAAP and other genes of interestcan be analyzed by northern analysis or microarray techniques.

Pseudouridine synthase activity of NAAP is assayed using a tritium (³H)release assay modified from Nurse et al. ((1995) RNA 1:102-112), whichmeasures the release of ³H from the C₅ position of the pyrimidinecomponent of uridylate (U) when ³H-radiolabeled U in RNA is isomerizedto pseudouridine (ψ). A typical 500 μl assay mixture contains 50 mMHEPES buffer (pH 7.5), 100 mM ammonium acetate, 5 mM dithiothreitol, 1mM EDTA, 30 units RNase inhibitor, and 0.1-4.2 μM [5-³H]tRNA(approximately 1 μCi/nmol tRNA). The reaction is initiated by theaddition of <5 μl of a concentrated solution of NAAP (or samplecontaining NAAP) and incubated for 5 min at 37° C. Portions of thereaction mixture are removed at various times (up to 30 min) followingthe addition of NAAP and quenched by dilution into 1 ml 0.1 M HClcontaining Norit-SA3 (12% w/v). The quenched reaction mixtures arecentrifuged for S min at maximum speed in a microcentrifuge, and thesupernatants are filtered through a plug of glass wool. The pellet iswashed twice by resuspension in 1 ml 0.1 M HCl, followed bycentrifugation. The supernatants from the washes are separately passedthrough the glass wool plug and combined with the original filtrate. Aportion of the combined filtrate is mixed with scintillation fluid (upto 10 ml) and counted using a scintillation counter. The amount of ³Hreleased from the RNA and present in the soluble filtrate isproportional to the amount of peudouridine synthase activity in thesample (Ramamurthy, V. (1999) J. Biol. Chem. 274:22225-22230).

In the alternative, pseudouridine synthase activity of NAAP is assayedat 30° C. to 37° C. in a mixture containing 100 mM Tris-HCl (pH 8.0),100 mM ammonium acetate, 5 mM MgCl₂, 2 mM dithiothreitol, 0.1 mM EDTA,and 1-2 fmol of [³²P]-radiolabeled runoff transcripts (generated invitro by an appropriate RNA polymerase, i.e., T7 or SP6) as substrates.NAAP is added to initiate the reaction or omitted from the reaction incontrol samples. Following incubation, the RNA is extracted withphenol-chloroform, precipitated in ethanol, and hydrolyzed completely to3-nucleotide monophosphates using RNase T₂. The hydrolysates areanalyzed by two-dimensional thin layer chromatography, and the amount of32p radiolabel present in the ψMP and UMP spots are evaluated afterexposing the thin layer chromatography plates to film or aPhosphorImager screen. Taking into account the relative number ofuridylate residues in the substrate RNA, the relative amount ψMP and UMPare determined and used to calculate the relative amount of ψ per tRNAmolecule (expressed in mol ψ/mol of tRNA or mol ψ/mol of tRNA/minute),which corresponds to the amount of pseudouridine synthase activity inthe NAAP sample (Lecointe, F. et al. (1998) J. Biol. Chem.273:1316-1323).

N²,N²-dimethylguanosine transferase ((m² ₂G)methyltransferase) activityof NAAP is measured in a 160 μl reaction mixture containing 100 mMTris-HCl (pH 7.5), 0.1 mM EDTA, 10 mM MgCl₂, 20 mM NH₄Cl, 1 mMdithiothreitol, 6.2 μM S-adenosyl-L-[methyl-³H]methionine (30-70 Ci/mM),8 μg m² ₂G-deficient tRNA or wild type tRNA from yeast, andapproximately 100 μg of purified NAAP or a sample comprising NAAP. Thereactions are incubated at 30° C. for 90 min and chilled on ice. Aportion of each reaction is diluted to 1 ml in water containing 100 μgBSA. 1 ml of 2 M HCl is added to each sample and the acid insolubleproducts are allowed to precipitate on ice for 20 min before beingcollected by filtration through glass fiber filters. The collectedmaterial is washed several times with HCl and quantitated using a liquidscintillation counter. The amount of ³H incorporated into the m²₂G-deficient, acid-insoluble tRNAs is proportional to the amount ofN²,N²-dimethylguanosine transferase activity in the NAAP sample.Reactions comprising no substrate tRNAs, or wild-type tRNAs that havealready been modified, serve as control reactions which should not yieldacid-insoluble ³H-labeled products.

Polyadenylation activity of NAAP is measured using an in vitropolyadenylation reaction. The reaction mixture is assembled on ice andcomprises 10 μl of 5 mM ditiothreitol, 0.025% (v/v) NONIDET P-40, 50 mMcreatine phosphate, 6.5% (w/v) polyvinyl alcohol, 0.5 unit/μl RNAGUARD(Pharmacia), 0.025 μg/μl creatine kinase, 1.25 mM cordycepin5′-triphosphate, and 3.75 mM MgCl₂, in a total volume of 25 μl. 60 fmolof CstF, 50 fmol of CPSF, 240 fmol of PAP, 4 μl of crude or partialpurified CF II and various amounts of amounts CF I are then added to thereaction mix. The volume is adjusted to 23.5 μl with a buffer containing50 mM TrisHCl, pH 7.9, 10% (v/v) glycerol, and 0.1 mM Na-EDTA. The finalammonium sulfate concentration should be below 20 mM. The reaction isinitiated (on ice) by the addition of 15 fmol of ³²P-labeled pre-mRNAtemplate, along with 2.5 μg of unlabeled tRNA, in 1.5 μl of water.Reactions are then incubated at 30° C. for 75-90 min and stopped by theaddition of 75 μl (approximately two-volumes) of proteinase K mix (0.2 MTris-HCl, pH 7.9, 300 mM NaCl, 25 mM Na-EDTA, 2% (w/v) SDS), 1 μl of 10mg/ml proteinase K, 0.25 μl of 20 mg/ml glycogen, and 23.75 μl ofwater). Following incubation, the RNA is precipitated with ethanol andanalyzed on a 6% (w/v) polyacrylamide, 8.3 M urea sequencing gel. Thedried gel is developed by autoradiography or using a phosphoimager.Cleavage activity is determined by comparing the amount of cleavageproduct to the amount of pre-mRNA template. The omission of any of thepolypeptide components of the reaction and substitution of NAAP isuseful for identifying the specific biological function of NAAP inpre-mRNA polyadenylation (Rüegsegger, U. et al. (1996) J. Biol. Chem.271:6107-6113; and references within).

tRNA synthetase activity is measured as the aminoacylation of asubstrate tRNA in the presence of [¹⁴C]-labeled amino acid. NAAP isincubated with [¹⁴C-labeled amino acid and the appropriate cognate tRNA(for example, [¹⁴C]alanine and tRNA^(ala)) in a buffered solution.¹⁴C-labeled product is separated from free [¹⁴C]amino acid bychromatography, and the incorporated ¹⁴C is quantified by scintillationcounter. The amount of ¹⁴C-labeled product detected is proportional tothe activity of NAAP in this assay.

In the alternative, NAAP activity is measured by incubating a samplecontaining NAAP in a solution containing 1 mM ATP, 5 mM Hepes-KOH (pH7.0), 2.5 mM KCl, 1.5 mM magnesium chloride, and 0.5 mM DTT along withmisacylated [¹⁴C]-Glu-tRNAGln (e.g., 1 μM) and a similar concentrationof unlabeled L-glutamine. Following the quenching of the reaction with 3M sodium acetate (pH 5.0), the mixture is extracted with an equal volumeof water-saturated phenol, and the aqueous and organic phases areseparated by centrifugation at 15,000×g at room temperature for 1 min.The aqueous phase is removed and precipitated with 3 volumes of ethanolat −70° C. for 15 min. The precipitated aminoacyl-tRNAs are recovered bycentrifugation at 15,000×g at 4° C. for 15 min. The pellet isresuspended in of 25 mM KOH, deacylated at 65° C. for 10 min.,neutralized with 0.1 M HCl (to final pH 6-7), and dried under vacuum.The dried pellet is resuspended in water and spotted onto a celluloseTLC plate. The plate is developed in either isopropanol/formicacid/water or ammonia/water/chloroform/methanol. The image is subjectedto densitometric analysis and the relative amounts of Glu and Gln arecalculated based on the Rf values and relative intensities of the spots.NAAP activity is calculated based on the amount of Gln resulting fromthe transformation of Glu while acylated as Glu-tRNA^(Gln) (adapted fromCurnow, A. W. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11819-26).

XIX. Identification of NAAP Agonists and Antagonists

Agonists or antagonists of NAAP activation or inhibition may be testedusing the assays described in section XVIII. Agonists cause an increasein NAAP activity and antagonists cause a decrease in NAAP activity.

XX. NAAP Secretion Assay

A high throughput assay may be used to identify polypeptides that aresecreted in eukaryotic cells. In an example of such an assay,polypeptide expression libraries are constructed by fusing 5′-biasedcDNAs to the 5′-end of a leaderless β-lactamase gene. β-lactamase is aconvenient genetic reporter as it provides a high signal-to-noise ratioagainst low endogenous background activity and retains activity uponfusion to other proteins. A dual promoter system allows the expressionof β-lactamase fusion polypeptides in bacteria or eukaryotic cells,using the lac or CMV promoter, respectively.

Libraries are first transformed into bacteria, e.g., E. coli, toidentify library members that encode fusion polypeptides capable ofbeing secreted in a prokaryotic system. Mammalian signal sequencesdirect the translocation of β-lactamase fusion polypeptides into theperiplasm of bacteria where it confers antibiotic resistance tocarbenicillin. Carbenicillin-selected bacteria are isolated on solidmedia, individual clones are grown in liquid media, and the resultingcultures are used to isolate library member plasmid DNA.

Mammalian cells, e.g., 293 cells, are seeded into 96-well tissue cultureplates at a density of about 40,000 cells/well in 100 μl phenol red-freeDME supplemented with 10% fetal bovine serum (FBS) (Life Technologies,Rockville, Md.). The following day, purified plasmid DNAs isolated fromcarbenicillin-resistant bacteria are diluted with 15 μl OPTI-MEM Imedium (Life Technologies) to a volume of 25 μl for each well of cellsto be transfected. In separate plates, 1 ;l LF2000 Reagent (LifeTechnologies) is diluted into 25 μl/well OPTI-MEM I. The 25 μl dilutedLF2000 Reagent is then combined with the 25 μl diluted DNA, mixedbriefly, and incubated for 20 minutes at room temperature. The resultingDNA-LF2000 reagent complexes are then added directly to each well of 293cells. Cells are also transfected with appropriate control plasmidsexpressing either wild-type β-lactamase, leaderless β-lactamase, or, forexample, CD4-fused leaderless β-lactamase. 24 hrs followingtransfection, about 90 μl of cell culture media are assayed at 37° C.with 100 μM Nitrocefin (Calbiochem, San Diego Calif.) and 0.5 mM oleicacid (Sigma, St. Louis, Mo.) in 10 mM phosphate buffer (pH 7.0).Nitrocefin is a substrate for β-lactamase that undergoes a noticeablecolor change from yellow to red upon hydrolysis. β-lactamase activity ismonitored over 20 min in a microtiter plate reader at 486 nm. Increasedcolor absorption at 486 nm corresponds to secretion of a β-lactamasefusion polypeptide in the transfected cell media, resulting from thepresence of a eukaryotic signal sequence in the fusion polypeptide.Polynucleotide sequence analysis of the corresponding library memberplasmid DNA is then used to identify the signal sequence-encoding cDNA.(Described in U.S. patent application Ser. No. 09/803,317, filed Mar. 9,2001.)

Various modifications and variations of the described compositions,methods, and systems of the invention will be apparent to those skilledin the art without departing from the scope and spirit of S theinvention. It will be appreciated that the invention provides novel anduseful proteins, and their encoding polynucleotides, which can be usedin the drug discovery process, as well as methods for using thesecompositions for the detection, diagnosis, and treatment of diseases andconditions. Although the invention has been described in connection withcertain embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments. 10Nor should the description of such embodiments be considered exhaustiveor limit the invention to the precise forms disclosed. Furthermore,elements from one embodiment can be readily recombined with elementsfrom one or more other embodiments. Such combinations can form a numberof embodiments within the scope of the invention. It is intended thatthe scope of the invention be defined by the following claims and theirequivalents. TABLE 1 Incyte Polypeptide Incyte PolynucleotidePolynucleotide Incyte Project ID SEQ ID NO: Polypeptide ID SEQ ID NO: ID7503848 1 7503848CD1 59 7503848CB1 2608080 2 2608080CD1 60 2608080CB17503402 3 7503402CD1 61 7503402CB1 7503517 4 7503517CD1 62 7503517CB17500014 5 7500014CD1 63 7500014CB1 7501365 6 7501365CD1 64 7501365CB17503540 7 7503540CD1 65 7503540CB1 7504326 8 7504326CD1 66 7504326CB17504388 9 7504388CD1 67 7504388CB1 2828380 10 2828380CD1 68 2828380CB16456919 11 6456919CD1 69 6456919CB1 7502244 12 7502244CD1 70 7502244CB17498718 13 7498718CD1 71 7498718CB1 6259308 14 6259308CD1 72 6259308CB17504104 15 7504104CD1 73 7504104CB1 7504121 16 7504121CD1 74 7504121CB15635695 17 5635695CD1 75 5635695CB1 7503983 18 7503983CD1 76 7503983CB17503476 19 7503476CD1 77 7503476CB1 7504023 20 7504023CD1 78 7504023CB17504128 21 7504128CD1 79 7504128CB1 4529338 22 4529338CD1 80 4529338CB17503460 23 7503460CD1 81 7503460CB1 5466630 24 5466630CD1 82 5466630CB17503474 25 7503474CD1 83 7503474CB1 7503498 26 7503498CD1 84 7503498CB17504119 27 7504119CD1 85 7504119CB1 71532805 28 71532805CD1 8671532805CB1 5502992 29 5502992CD1 87 5502992CB1 7503828 30 7503828CD1 887503828CB1 2647325 31 2647325CD1 89 2647325CB1 7495416 32 7495416CD1 907495416CB1 8096177 33 8096177CD1 91 8096177CB1 666763 34 666763CD1 92666763CB1 7504091 35 7504091CD1 93 7504091CB1 7503568 36 7503568CD1 947503568CB1 7504101 37 7504101CD1 95 7504101CB1 6946680 38 6946680CD1 966946680CB1 7001142 39 7001142CD1 97 7001142CB1 71158380 40 71158380CD198 71158380CB1 7503861 41 7503861CD1 99 7503861CB1 7758395 42 7758395CD1100 7758395CB1 71039312 43 71039312CD1 101 71039312CB1 7291318 447291318CD1 102 7291318CB1 2638619 45 2638619CD1 103 2638619CB1 281001446 2810014CD1 104 2810014CB1 3457155 47 3457155CD1 105 3457155CB17435171 48 7435171CD1 106 7435171CB1 7499936 49 7499936CD1 1077499936CB1 7504125 50 7504125CD1 108 7504125CB1 7505742 51 7505742CD1109 7505742CB1 7505757 52 7505757CD1 110 7505757CB1 7504126 537504126CD1 111 7504126CB1 7504099 54 7504099CD1 112 7504099CB1 750573355 7505733CD1 113 7505733CB1 7959829 56 7959829CD1 114 7959829CB17502168 57 7502168CD1 115 7502168CB1 7503888 58 7503888CD1 1167503888CB1 Incyte Polypeptide Incyte Polynucleotide PolynucleotideIncyte Project ID SEQ ID NO: Polypeptide ID SEQ ID NO: ID Incyte FullLength Clones 7503848 1 7503848CD1 59 7503848CB1 2608080 2 2608080CD1 602608080CB1 7503402 3 7503402CD1 61 7503402CB1 6308169CA2 7503517 47503517CD1 62 7503517CB1 7500014 5 7500014CD1 63 7500014CB1 90040096CA2,90045149CA2, 90045157CA2, 90045165CA2, 90045181CA2, 90045189CA2,90045201CA2, 90045233CA2, 90045249CA2, 90045265CA2, 90045273CA2,90045281CA2, 90045289CA2, 90166707CA2, 90166739CA2, 90166815CA2,90166831CA2 7501365 6 7501365CD1 64 7501365CB1 7503540 7 7503540CD1 657503540CB1 7504326 8 7504326CD1 66 7504326CB1 7504388 9 7504388CD1 677504388CB1 2828380 10 2828380CD1 68 2828380CB1 6456919 11 6456919CD1 696456919CB1 3212008CA2 7502244 12 7502244CD1 70 7502244CB1 7498718 137498718CD1 71 7498718CB1 6259308 14 6259308CD1 72 6259308CB1 8653345CA27504104 15 7504104CD1 73 7504104CB1 2654926CA2 7504121 16 7504121CD1 747504121CB1 5635695 17 5635695CD1 75 5635695CB1 7503983 18 7503983CD1 767503983CB1 2215488CA2, 8662527CA2 7503476 19 7503476CD1 77 7503476CB17504023 20 7504023CD1 78 7504023CB1 7504128 21 7504128CD1 79 7504128CB14529338 22 4529338CD1 80 4529338CB1 7503460 23 7503460CD1 81 7503460CB190062547CA2, 90062615CA2, 90062623CA2, 90062639CA2 5466630 24 5466630CD182 5466630CB1 7503474 25 7503474CD1 83 7503474CB1 7503498 26 7503498CD184 7503498CB1 2170945CA2 7504119 27 7504119CD1 85 7504119CB1 95135029CA271532805 28 71532805CD1 86 71532805CB1 5502992 29 5502992CD1 875502992CB1 7503828 30 7503828CD1 88 7503828CB1 2647325 31 2647325CD1 892647325CB1 90177208CA2 7495416 32 7495416CD1 90 7495416CB1 8096177 338096177CD1 91 8096177CB1 90170506CA2 666763 34 666763CD1 92 666763CB17504091 35 7504091CD1 93 7504091CB1 7503568 36 7503568CD1 94 7503568CB17504101 37 7504101CD1 95 7504101CB1 6946680 38 6946680CD1 96 6946680CB17001142 39 7001142CD1 97 7001142CB1 90180809CA2 71158380 40 71158380CD198 71158380CB1 4913234CA2 7503861 41 7503861CD1 99 7503861CB1 7758395 427758395CD1 100 7758395CB1 71039312 43 71039312CD1 101 71039312CB17291318 44 7291318CD1 102 7291318CB1 2638619 45 2638619CD1 1032638619CB1 2810014 46 2810014CD1 104 2810014CB1 3387728CA2, 90166951CA2,90166967CA2, 90166975CA2, 90166983CA2, 90166991CA2, 90167051CA2,90167067CA2 3457155 47 3457155CD1 105 3457155CB1 7435171 48 7435171CD1106 7435171CB1 7499936 49 7499936CD1 107 7499936CB1 90041227CA2,90041243CA2, 90041319CA2 7504125 50 7504125CD1 108 7504125CB190057593CA2, 90057785CA2, 90057853CA2, 90057955CA2, 90057963CA2,90057971CA2, 90057979CA2, 90057987CA2, 90057995CA2, 90058033CA2,90058055CA2, 90058063CA2, 90058071CA2, 90058079CA2, 90058087CA2,90058095CA2 7505742 51 7505742CD1 109 7505742CB1 7505757 52 7505757CD1110 7505757CB1 7504126 53 7504126CD1 111 7504126CB1 4549855CA2 750409954 7504099CD1 112 7504099CB1 7505733 55 7505733CD1 113 7505733CB17959829 56 7959829CD1 114 7959829CB1 4111545CA2, 90176769CA2,90176777CA2, 90176785CA2, 90176853CA2, 90176861CA2, 90176869CA2 750216857 7502168CD1 115 7502168CB1 7503888 58 7503888CD1 116 7503888CB1

TABLE 2 Poly- peptide GenBank ID NO: SEQ Incyte or PROTEOME ProbabilityID NO: Polypeptide ID ID NO: Score Annotation 1 7503848CD1 g1854952 0.0[Homo sapiens] putative nucleolar trafficking phosphoprotein Wise, C. A.et al. (1997) TCOF1 gene encodes a putative nucleolar phosphoproteinthat exhibits mutations in Treacher Collins Syndrome throughout itscoding region. Proc. Natl. Acad. Sci. U.S.A. 94: 3110-3115 338442|TCOF10.0 [Homo sapiens][Nuclear import/exportprotein; Transporter] [Nuclearnucleolus; Nuclear] Treacle, protein with similarity to nucleolartrafficking proteins that isphosphorylated by casein kinase; mutation ofcorresponding genecauses Treacher Collins Syndrome 320096|Tcof1 2.7E−211[Mus musculus][Nuclear import/export protein] [Nuclear nucleolus;Nuclear] Protein with similarity to nucleolar phosphoproteins, may havea role in nucleolar- cytoplasmic transportand craniofacial development;putative human ortholog TCOF1 is associated with Treacher CollinsSyndrome 239850|C25A1.10 6.1E−41 [Caenorhabditis elegans][Nuclearimport/exportprotein][Nuclear pore] Putative nucleoporin, has moderatesimilarity to H. sapeins P130 gene product [nucleolar phosphoproteinp130] 247598|K06A9.1 2.4E−35 [Caenorhabditis elegans] Putative mucin,has strong similarity to H. sapiens MUC1 gene product [mucin 1,transmembrane] 630082|orf6.162 2.8E−31 [Candida albicans] Protein ofunknown function, has a region of low similarity to C. albicans Hwp1p,which is a hyphal-specific cell wall protein with a role in attachmentto host epithelial cells 2 2608080CD1 g1020145 1.1E−149 [Homo sapiens]DNA binding protein Bellefroid, E. J. et al. (1989) The human genomecontains hundreds of genes coding for finger proteins of the Kruppeltype. DNA 8: 377-387 346272|ZNF264 3.7E−182 [Homo sapiens][Inhibitor orrepressor; Transcription factor] Protein with high similarity to ZNF184,which is a KRAB zinc finger protein that is expressed in testis,contains a KRAB (kruppel-associated box) domain, which may mediatetranscriptional repression, and twelve C2H2 type zinc finger domains339004|ZNF84 9.4E−151 [Homo sapiens][Inhibitor or repressor; DNA-bindingprotein; Transcription factor] [Nuclear] Protein containing a KRAB(kruppel-associated box) domain which may mediate transcriptionalrepression and several C2H2 type zinc finger domains, which bind nucleicacids 308339|ZNF184 2.7E−149 [Homo sapiens] Kruppel-like zinc-fingerprotein, maximally expressed in testis, moderately in other tissues339006|ZNF85 2.9E−145 [Homo sapiens][Inhibitor or repressor;Transcription factor; DNA-binding protein] [Nuclear] Zinc-fingertranscriptional repressor containing a Kruppel-associated box (KRAB)domain, member of the ZNF91 family of zinc-finger proteins 339008|ZNF917.8E−145 [Homo sapiens] Zinc-finger protein containing aKruppel-associated box (KRAB) transcriptional repression domain, mosthighly expressed in T lymphoid cells and down-regulated during in vitroterminal differentiation of myeloid cells 3 7503402CD1 g495572 0.0 [Homosapiens] zinc finger protein Tommerup, N. and Vissing, H. (1995)Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAsidentify putative candidate genes for developmental and malignantdisorders. Genomics 27: 259-264 338964|ZNF143 0.0 [Homo sapiens][Activator; DNA-binding protein; Transcription factor] Zinc- fingertranscriptional activator of small nuclear (snRNA) and snRNA-type genestranscribed by RNA polymerases II and III 324316|D7Ertd805e 0.0 [Musmusculus][Activator; DNA-binding protein; Transcription factor] Zinc-finger transcriptional activator of the selenocysteine tRNA (tRNAsec),binding activity in mammary glands increases in parallel with theincrease of tRNAsec transcript during the periods of pregnancy andlactation 339000|ZNF76 1.9E−130 [Homo sapiens][Activator; Transcriptionfactor; DNA-binding protein] Kruppel- like zinc-finger transcriptionalactivator of the small nuclear (snRNA) and snRNA- type genes transcribedby RNA polymerases II and III, expressed in testis 324156|Mm.105091.4E−54 [Mus musculus][DNA-binding protein][Nuclear] Protein containinga C2H2 type zinc finger domain, which bind nucleic acids 432838|ZNF1801.6E−54 [Homo sapiens] Zinc finger protein; corresponding gene islocalized in a region associated with rearrangements leading todevelopmental abnormalities, DNA repair deficiencies, and cellularmalignancies 4 7503517CD1 g9651997 4.7E−219 [Homo sapiens] eukaryotictranslation initiation factor EIF2B subunit 3 Kruger, M. et al. (2000)Identification of eIF2B gamma and eIF2 gamma as cofactors of hepatitis Cvirus internal ribosome entry site-mediated translation using afunctional genomics approach. Proc. Natl. Acad. Sci. U.S.A. 97:8566-8571 610840|EIF2B3 4.1E−220 [Homo sapiens][Translationfactor][Cytoplasmic] Subunit of eukaryotic translation initiation factor2B 330762|Rn.10577 1.9E−200 [Rattus norvegicus][Guanine nucleotideexchange actor; Translation factor][Cytoplasmic] Gamma subunit oftranslation initiation factor 2B, a heteropentamer that mediates theexchange of GDP bound to translation initiation factor eIF2 for GTP439325|ppp-1 8.8E−39 [Caenorhabditis elegans] [Transferase; Translationfactor] [Cytoplasmic] Protein containing a putative NTP transferase(nucleotidyl transferase) domain, has weak similarity to S. cerevisiaePsa1p (mannose-1-phosphate guanyltransferase; GDP- mannosepyrophosphorylase) 370068|tif223 1.1E−33 [Schizosaccharomyces pombe][Translation factor] Putative translation initiation factor eIF-2b gammasubunit, has low similarity to S. cerevisiae Gcd1p 643938|orf6.70906.8E−20 [Candida albicans][Guanine nucleotide exchange factor;Translation factor] Protein containing three bacterial transferasehexapeptide (four repeats) domains, has low similarityto S. cerevisiaeGcd1p, which is a translation initiation factor eIF2B 5 7500014CD1g12654757 1.6E−55 [Homo sapiens] nuclear receptor binding protein432864|NRBP 1.4E−56 [Homo sapiens][Nuclear] Adaptor protein with twonuclear receptor binding motifs, a SH2 binding domain, a kinase-likedomain and a nuclear localization signal, may have a role in thesignaling pathways involving nuclear receptors and SH2 domain containingproteins 6 7501365CD1 g11322247 3.9E−210 [Homo sapiens] nucleolarprotein No55 343772|SC65 9.2E−211 [Homo sapiens][Nuclear nucleolus;Nuclear] Nucleolar protein that associates with chromosomes duringmitosis and has similarityto rat SC65 (Rn.40377), a synaptonemal complexprotein 333646|Sc65 2.0E−176 [Rattus norvegicus][DNA-bindingprotein][Nuclear] Component of synaptonemal complex localized betweenpaired aligned cores of homologous chromosomes 609086|Crtap 1.3E−110[Mus musculus] Cartilage associated protein, a protein that is expressedin embryonic cartilage 343398|CRTAP 8.9E−110 [Homo sapiens] Cartilageassociated protein, has strong similarity to murine Crtap, which is aprotein that is expressed in embryonic cartilage 613744|Gros1 7.5E−50[Mus musculus] [Inhibitor or repressor] Growth suppressor, expression incell culture results in slow growth of cells and reducedcolony-formation 7 7503540CD1 g5734605 0.0 [Homo sapiens] KARP-1-bindingprotein 3 346328|KIAA0470 0.0 [Homo sapiens] Protein containing aforkhead associated (FHA), which bind phosphotyrosine residues434396|KIAA0284 1.5E−125 [Homo sapiens] Protein of unknown function, hasa region of low similarity to a region of rat Rn.32072, which is asalivary protein belonging to a proline-rich protein family that alsoincludes RP13 (Rn.9841) and RP15 (Rn.9842) 4988|MUC1 6.9E−12[Saccharomyces cerevisiae] [Hydrolase] [Cell wall] Cellsurfaceflocculin, required for invasive and pseudohyphal growth 87504326CD1 g14915787 0.0 [Mus musculus] WAC 4988|MUC1 1.9E−15[Saccharomyces cerevisiae] [Hydrolase] [Cell wall] Cell surfaceflocculin, required for invasive and pseudohyphal growth370430|SPBC215.13 3.5E−11 [Schizosaccharomyces pombe] Serine-richprotein 9 7504388CD1 g14009498 4.8E−86 [Homo sapiens] hairy/enhancer ofsplit 6 Vasiliauskas, D. and Stern, C. D. (2000) Expression of mouseHES-6, a new member of the Hairy/Enhancer of split family of bHLHtranscription factors. Mech. Dev. 98: 133-137 599700|HES6 1.4E−100 [Homosapiens][Inhibitor or repressor; Transcription factor] Basic helix-loop-helix protein, does not bind DNA but acts as an inhibitor of Hes1 andsuppresses Hes1 from repressing transcription 608436|Hes6 4.5E−85 [Musmusculus] Member of the family of homologs of Drosophila hairy andEnhancer of split, a basic helix-loop-helix protein that inhibits thetranscriptional repressor Hes1 and promotes cell differentiation321888|Hes1 5.7E−14 [Mus musculus][Inhibitor or repressor; DNA-bindingprotein; Transcription factor] Hairy and enhancer of split, ahelix-loop-helix negative regulator of transcription 344428|HRY 7.3E−14[Homo sapiens][DNA-binding protein] Homolog of Drosophila hairy, hasvery strong similarity to murine Hes1, which is a helix-loop-helixnegative regulator of transcription, has very strong similarity to ratRn.19727, which suppresses neuronal differentiation 688984|Hes1 9.8E−14[Rattus norvegicus] [Inhibitor or repressor; DNA-binding protein;Transcription factor] [Nuclear] Hairy-like, transduces growth factorsignals during embryonic development 10 2828380CD1 g13752754 8.5E−240[Homo sapiens] zinc finger 1111 339008|ZNF91 9.9E−230 [Homo sapiens]Zinc-finger protein 91 (HPF7, HTF10), member of KRAB subfamily of C2H2zinc finger proteins, functions as a transcriptional repressor, may playa role in formation of seminomas, down-regulated during in vitro myeloidcell differentiation 691254|FLJ14345 6.6E−217 [Homo sapiens] Proteinwith high similarity to human ZNF255, which is kruppel- like zinc fingerprotein that may activate transcription 432896|ZNF208 2.6E−213 [Homosapiens] Zinc finger protein 208, a ubiquitously expressed Kruppel-associated box (KRAB) zinc finger protein 338994|ZNF43 2.3E−205 [Homosapiens] Zinc finger protein 43, contains C2H2 zinc finger motifs,expressed mainly in B and T cells 365207|ZNF197 1.1E−200 [Homosapiens][Transcription factor] Zinc finger protein 197, member of thezinc- finger transcription factor family, contains twenty C2H2-type zincfinger motifs, high level expressionis associated with thyroid papillarycarcinomas 11 6456919CD1 g930123 1.7E−147 [Homo sapiens] zinc fingerprotein (583 AA) 435298|ZNF20 7.3E−163 [Homo sapiens][DNA-bindingprotein; Transcription factor; Small molecule- binding protein] PutativeDNA-binding protein with a zinc finger motif 594469|HSZFP36 1.5E−148[Homo sapiens][Inhibitor or repressor; Transcription factor; DNA-bindingprotein] Protein containing fourteen C2H2 type zinc finger domains,which bind nucleic acids, also contains a KRAB (kruppel-associated box)domain which may mediate transcriptional repression 623668|ZNF143.1E−146 [Homo sapiens] Zinc finger protein isolated from cell lines ofT-cell origin 476345|LOC51712 3.1E−146 [Homo sapiens][Inhibitor orrepressor; Transcription factor; DNA-binding protein] Protein containingeighteen C2H2 type zinc finger domains, which bind nucleic acids, alsocontains a KRAB (kruppel-associated box) domain which may mediatetranscriptional repression 338956|ZNF136 7.5E−145 [Homosapiens][Inhibitor or repressor; Transcriptionfactor; DNA-bindingprotein] C2H2 zinc-finger protein containing a Kruppel-associated box-A(KRAB-A) transcriptional repression domain, represses transcription whenfused to the heterologous KRABB subdomain of human ZNF10 13 7498718CD1g2897601 7.4E−170 [Homo sapiens] kruppel-type zinc finger protein Blin,N. (1997) Expressed sequences within pericentromeric heterochromatin ofhuman chromosome 22. Mamm. Genome 8: 859-862 704009|ZNF73 6.4E−171 [Homosapiens] Member of the Kruppel type family of zinc finger proteins338968|ZNF157 1.0E−123 [Homo sapiens][Inhibitor or repressor;Transcription factor] Zinc finger protein 157, a zinc-finger proteinthat contains two Kruppel-associated box (KRAB-A and KRAB-B)transcription repression domains 308339|ZNF184 4.5E−123 [Homo sapiens]Kruppel-like zinc-finger protein, maximally expressed in testis,moderately in other tissues 435075|ZNF41 1.4E−121 [Homosapiens][Inhibitor or repressor; Transcription factor] Zinc fingerprotein with 18 contiguous zinc fingers of the C2H2 type, contains aKRAB/FPB (Kruppel-associated/finger preceding box) domain, whichprobably functions in transcriptional repression 587437|Zfp68 7.6E−120[Mus musculus][Inhibitor or repressor; Transcription factor]KRAB-containing zinc-finger protein that when bound to the corepressorKAP-1, forms a functional transcriptional repressor complex 146259308CD1 g1916290 4.0E−130 [Mus musculus] ALY Bruhn, L. (1997) ALY, acontext-dependent coactivator of LEF-1 and AML-1, is required forTCRalpha enhancer function. Genes Dev. 11: 640-653 585985|Refbp13.5E−131 [Mus musculus][Transcription factor; RNA-binding protein][Nuclear; Cytoplasmic] Member of the T-cell receptor alpha (TCR alpha)enhancer complex that interacts with the activation domains of LEF-1 andAML-1 to stimulate transcription from theT-cell receptor alpha (TCRalpha) enhancer 348154|ALY 1.1E−120 [Homo sapiens][Activator;DNA-binding protein; Transcriptionfactor; RNA- binding protein][Nuclear] Ortholog of murine Mm. 1886, a member of the T-cell receptoralpha (TCR alpha) enhancer complex that acts to stimulate transcriptionfrom the T-cell receptor alpha (TCR alpha) enhancer, may have a role insystemic lupus erythematosus 597373|Refbp2 1.6E−99 [Musmusculus][RNA-binding protein] [Nuclear] RNA and expor tactor bindingprotein 2, member of a conserved family of heterogeneous nuclearribonucleoprotein-like proteins which binds nuclear RNA and has a rolein mRNA export from the nucleus, contains an RNA recognition motif (RRM)domain 243563|F23B2.6 2.9E−19 [Caenorhabditis elegans][RNA-bindingprotein] Member of the RRM domain protein family 239659|C18D11.4 3.0E−16[Caenorhabditis elegans][RNA-binding protein] [Nuclear] Protein withstrong similarity to human SFRS10 protein and SR-like splicing factorand Drosophila TRA2, (putative RNA binding protein) 15 7504104CD1g338013 4.5E−266 [Homo sapiens] SEF2-1A protein Corneliussen, B. (1991)Helix-loop-helix transcriptional activators bind to a sequence inglucocorticoid response elements of retrovirus enhancers. J. Virol. 65:6084-6093 338432|TCF4 1.2E−263 [Homo sapiens][Activator; DNA-bindingprotein; Transcription factor] [Nuclear] Transcription factor 4, basichelix-loop-helix transcriptional co-activator and repressor, plays arole in the Wnt signaling pathway; mutations in the corresponding geneare associated with colorectal tumors 587379|Tcf4 5.3E−263 [Musmusculus][Activator; Inhibitor or repressor; DNA-binding protein;Transcription factor; Small molecule-binding protein] Transcriptionfactor 4, basic helix-loop-helix transcriptional co-activator andco-repressor, plays a role in the Wnt signaling pathway and is essentialfor normal gastrulation; mutations in the human TCF4 gene are associatedwith colorectal tumors 330540|Rn.10450 2.1E−229 [Rattusnorvegicus][Activator; DNA-binding protein; Transcription factor][Nuclear] Transcription factor 4, hepatocyte nuclear factor 4 alpha,basic helix- loop-helix transcriptional co-activator and repressor,activates beta-cell genes involved in glucose metabolism; mutations inthe human TCF4 gene are associated with colorectal tumors 339804|TCF127.8E−166 [Homo sapiens][Activator; DNA-binding protein; Transcriptionfactor] Basic helix- loop-helix (bHLH) transcriptional activator thatbinds to the immunoglobulin enhancer E-box consensus sequence, formscomplexes with the immunoglobulin enhancer binding proteins E12 and ITF2and the myogenic factor myogenin (MYOG) 330280|Rn.10290 2.1E−91 [Rattusnorvegicus][Activator; Transcription factor; DNA-binding protein]Transcriptional activator with similarity to E12 and E47, may beinvolved in the regulation of pancreatic exocrine genes, includinginsulin and chymotrypsin 16 7504121CD1 g3258665 9.8E−191 [Gallus gallus]transcription factor LEF-1 Kengaku, M. (1998) Distinct WNT pathwaysregulating AER formation and dorsoventral polarity in the chick limbbud. Science 280: 1274-1277 7504121CD1 625410|LEF1 7.6E−120 [Homosapiens][Activator; Inhibitor or repressor; DNA-binding protein;Transcription factor] [Nuclear] Protein with very strong similarity tomurine Lef1, which is a member of the HMG-box family of transcriptionfactors that activates transcription of T-cell receptor alpha (TCRA),and which may regulate lymphocyte gene expression and differentiation585197|Lef1 6.6E−82 [Mus musculus][Activator; DNA-binding protein;Transcription factor] [Nuclear] Lymphoid enhancer binding factor 1,member of the HMG-box family of transcription factors, activatestranscription of T-cell receptor alpha (TCRA), may be a regulator oflymphocyte gene expression and differentiation 338436|TCF7 3.8E−63 [Homosapiens][Activator; DNA-binding protein; Transcription factor]Transcription factor 7, transcriptional activator that binds to Tcell-specific elements and plays a role in T cell differentiation; maybe associated with late events in colorectal cell tumor progression429226|Tcf7 2.7E−60 [Mus musculus][Activator; DNA-binding protein;Transcription factor] Transcription factor 7, transcriptional activatorthat binds to T cell-specific elements and plays a role in T celldifferentiation; human TCF7 may be associated with late events incolorectal cell tumor progression 429228|Tcf712 2.8E−58 [Musmusculus][Activator; DNA-binding protein; Transcription factor][Nuclear] HMG-box transcriptional activator, forms a complex withbeta-catenin (Catnb) or Armadillo that stimulates transcription inresponse to Wnt/Wingless signaling, may be involved in gastrointestinaltract development 17 5635695CD1 g14333988 0.0 [Homo sapiens] enhancer ofpolycomb 1 697396|EPC1 2.8E−200 [Homo sapiens] Enhancer of polycomb,both represses and activates transcription 325698|Epc1 1.2E−192 [Musmusculus] Protein with similarity to the Drosophila enhancer of polycombE(PC) gene, may regulate chromatin structure 256495|Y111B2 5.3E−51[Caenorhabditis elegans] Protein with strong similarity to D.melanogaster E(Pc) A.I (Enhancer of Polycomb) protein 18 7503983CD1g15215451 4.8E−132 [Homo sapiens] (BC012819) eukaryotic translationelongation factor 1 delta (guanine nucleotide exchange protein)742632|FLJ20897 4.3E−129 [Homo sapiens] Translation elongation factor 1delta, a guanine-nucleotide exchange protein that contains a leucinezipper motif 742436|EEF1B2 2.3E−56 [Homo sapiens] Eukaryotic translationelongation factor 1beta 2, putative component of the eukaryotictranslation elongation complex 608120|Eef1b2 1.6E−55 [Mus musculus][Guanine nucleotide exchange factor; Translation factor] [Cytoplasmic]Protein with very strong similarity to human EEF1B2, eukaryotictranslation elongation factor 1 beta 2, a putative component of theeukaryotic translation elongation complex 276349|F54H12.6 3.3E−46[Caenorhabditis elegans] Member of the elongation factor 1 (beta/deltachain) protein family 252376|Y41E3.10 3.4E−46 [Caenorhabditis elegans][Translation factor] [Cytoplasmic] Putative translation elongationfactor 1[beta/delta chain] 19 7503476CD1 g550017 1.4E−31 [Homo sapiens]ribosomal protein L27a 337714|RPL27A 1.2E−32 [Homo sapiens] [Structuralprotein; RNA-binding protein; Ribosomal subunit] [Cytoplasmic] Ribosomalprotein L27a, component of the large 60S ribosomal subunit; gene isabnormally expressed in colorectal carcinomas Belhumeur, P. et al.(1987) Nucleic Acids Res 15: 1019-1029 Isolation and characterisation ofa murine cDNA clone highly homologous to the yeast L29 ribosomal proteingene. 674449|Rpl27a 8.8E−32 [Mus musculus] [Structural protein;RNA-binding protein; Ribosomal subunit] [Cytoplasmic] Ribosomal proteinL27a, component of the large 60S ribosomal subunit; human RPL27A isabnormally expressed in colorectal carcinomas 6726|RPL28 1.2E−18[Saccharomyces cerevisiae] [RNA-binding protein; Ribosomal subunit][Nuclear; Cytoplasmic] Ribosomal protein L28 (yeast L29; YL24; rp44;mouse and rat L27a) 371142|rpl28-2 6.7E−18 [Schizosaccharomyces pombe][Ribosomal subunit] 60S ribosomal protein L28B/L27a/L29 376062|rpl28-11.1E−17 [Schizosaccharomyces pombe] [Ribosomal subunit] 60S ribosomalprotein L28 20 7504023CD1 g12653155 9.3E−102 [Homo sapiens] ribosomalprotein, large, P0 337756|RPLP0 8.1E−103 [Homo sapiens] [Structuralprotein; RNA-binding protein; Ribosomal subunit] [Cytoplasmic] Ribosomalprotein P0, acidic phosphoprotein component of the large 60S ribosomalsubunit; shows increased expression in hepatocellular and coloncarcinomas Krowczynska, A. M. et al. (1989) Nucleic Acids Res. 17: 6408The mouse homologue of the human acidic ribosomal phosphoprotein PO: ahighly conserved polypeptide that is under translational control.327804|Rn.1079 2.1E−102 [Rattus norvegicus] [Structural protein;RNA-binding protein; Ribosomal subunit] [Cytoplasmic] Ribosomal proteinP0, acidic phosphoprotein component of the large 60S ribosomal subunit;human RPLP0 shows increased expression in human hepatocellular and coloncarcinomas 580899|Arbp 0.0 [Mus musculus] [Structural protein;RNA-binding protein; Ribosomal subunit] [Cytoplasmic] Ribosomal proteinP0, acidic phosphoprotein component of the large 60S ribosomal subunit;human RPLP0 shows increased expression in human hepatocellular and coloncarcinomas 243695|F25H2.10 4.8E−53 [Caenorhabditis elegans] [Complexassembly protein] [Cytoplasmic] Ortholog of S. cerevisiae ribosomalprotein Rpp0p and member of the acidic ribosomal protein family370906|rpp0 3.7E−47 [Schizosaccharomyces pombe] [Ribosomal subunit] 60Sacidic ribosomal protein P0 21 7504128CD1 g186800 2.3E−71 [Homo sapiens]ribosomal protein L12 Chu, W. et al. (1993) Nucleic Acids Res. 21:749-749 The primary structure of human ribosomal protein L12337686|RPL12 2.0E−72 [Homo sapiens] [Structural protein; RNA-bindingprotein; Ribosomal subunit] [Cytoplasmic] Ribosomal protein L12,component of the large 60S ribosomal subunit 429164|Rpl12 6.8E−72 [Musmusculus] [Structural protein; RNA-binding protein; Ribosomal subunit][Cytoplasmic] Ribosomal protein L12, a component of the 60S ribosomalsubunit 247161|rpl-12 1.6E−54 [Caenorhabditis elegans] [RNA-bindingprotein] [Cytoplasmic] Member of the ribosomal protein L12 proteinfamily 371019|rpl12-1 1.4E−38 [Schizosaccharomyces pombe] [Ribosomalsubunit] 60S ribosomal protein L12A, has high similarity to S.cerevisiae Rpl12ap and S. cerevisiae Rpl12bp 370868|rpl12-2 1.4E−38[Schizosaccharomyces pombe] [Ribosomal subunit] 60S ribosomal proteinL12B, has high similarity to S. cerevisiae Rpl12ap and S. cerevisiaeRpl12bp 22 4529338CD1 g7542351 1.9E−187 [Homo sapiens] QUAKING isoform 6658120|QKI 1.6E−188 [Homo sapiens] [RNA-binding protein] Protein withvery strong similarity to murine qk, which is a putative RNA-bindingprotein that functions during embryonic myelination; mutations in themurine gene have effects ranging from embryonic death to quaking due todemyelination Ebersole, T. A. et al. (1996) Nat Genet 12: 260-265 Thequaking gene product necessary in embryogenesis and myelination combinesfeatures of RNA binding and signal transduction proteins. 626514|qk6.7E−160 [Mus musculus] [RNA-binding protein] Putative RNA-bindingprotein that has a role in myelination during embryogenesis; mutationsofthe corresponding gene have effects ranging from embryonic death to atransient quaking phenotype caused by demyelination 250880|T21G5.51.8E−65 [Caenorhabditis elegans] Putative paralog of C. elegans GLD-1which encodes an RNA-binding protein required for transition frommitosis to meiosis during spermatogenesis and oogenesis inhermaphrodites 251086|gld-1 5.0E−61 [Caenorhabditis elegans] [Inhibitoror repressor; RNA-binding protein] [Cytoplasmic] RNA binding protein,required for transition from mitosis to meiosis during spermatogenesisand oogenesis in hermaphrodites 364843|T-STAR 5.2E−34 [Homo sapiens][RNA-binding protein; Small molecule-binding protein] [Nuclear]RNA-binding protein that has similarity to theSrc associated SAM68protein, interacts with the testis-specific RBM RNA-binding protein andis expressed primarily in the testis 23 7503460CD1 g899298 2.8E−67 [Homosapiens] human splicing factor Kramer, A. et al. (1995) RNA 1: 260-272Mammalian splicing factor SF3a120 represents a new member of the SURPfamily of proteins and is homologous to the essential splicing factorPRP21p of Saccharomyces cerevisiae 742382|SF3A1 2.4E−68 [Homo sapiens]Splicing factor 3a subunit 1, component of histone deacetylasecomplexes, may be involved in transcriptional repression 251943|prp-212.0E−23 [Caenorhabditis elegans] [RNA-binding protein] [Nuclear]Putative U2 snRNP- associated splicing factor, putative ortholog ofhuman SAP114/SF3a120 and yeast Prp21p, member of the SWAP protein family369888|sap114 1.3E−21 [Schizosaccharomyces pombe] Pre-mRNA splicingfactor 341234|SFRS8 6.8E−15 [Homo sapiens] [Spliceosomal subunit;RNA-binding protein] [Nuclear] Splicing factor arginine serine rich 8, amemberof the SR protein family, regulates alternative splicing byinfluencing the selection of alternative 5′ splice sites, affectsalternative splicing of fibronectin, CD45 (PTPRC), and its own mRNA639178|orf6.4710 5.0E−10 [Candida albicans] Protein containing twoSurpmodules (SWAP domain) which may mediate RNA binding, has lowsimilarity to a region of human SF3A120 protein, which is the largesubunit (p120) of the SF3A splicing factor and involved in activation ofU2 snRNP 24 5466630CD1 g7290296 1.8E−239 [Drosophila melanogaster] kzgene product 276103|C06E1.10 6.9E−206 [Caenorhabditis elegans][Helicase] Member of the RNAhelicase, DEAH-box protein family 1295|ECM169.7E−161 [Saccharomyces cerevisiae] [Hydrolase; helicase; RNA-bindingprotein] [Nuclear nucleolus; Nuclear] Putative DEAH-box RNA helicase,directly implicated in ribosome biogenesis Lussier, M. et al. (1997)Genetics 147: 435-450 Large scale identification of genes involved incell surface biosynthesis and architecture in Saccharomyces cerevisiae.657990|SPAPB1 2.8E−155 [Schizosaccharomyces pombe] [Nuclear nucleolus]Putative ATP-dependent RNA A10.06c helicase 644688|orf6.7465 2.9E−119[Candida albicans] [RNA-binding protein] Protein with high similarity toS. cerevisiae Ecm16p, which is a putative DEAH-box RNA helicase directlyimplicated in ribosome biogenesis, contains a helicase conservedC-terminal domain 371297|prp22 1.8E−91 [Schizosaccharomyces pombe]Putative pre-mRNA splicing factor ATP- dependent RNA helicase 257503474CD1 g7243749 2.7E−124 [Homo sapiens] sir2-related protein type 6Frye, R.A. (2000) Biochem. Biophys. Res. Commun. 273: 793-798Phylogenetic classification of prokaryotic and eukaryotic Sir2-likeproteins 476521|SIRT6 2.4E−125 [Homo sapiens] Protein with lowsimilarity to SIRT3 and SIRT4, which are putativeADP-ribosyltransferases, and to members of the Sir2p family oftranscriptional regulatory proteins 476523|SIRT7 4.9E−29 [Homo sapiens]Protein with low similarity to members of the Sir2p family oftranscriptional regulatory proteins 724128|1ici_A 3.6E−11 [Protein DataBank] Transcriptional Regulatory Protein, Sir2 Fam 373770|SPCC132.023.8E−10 [Schizosaccharomyces pombe] [Transferase] Protein with highsimilarity to human SIRT2, which is a putative NAD-dependent deacetylaseand ADP- ribosyltransferase, member of the Sir2 family, which are silentinformation regulators 642492|orf6.6367 2.4E−09 [Candida albicans]Member of the Sir2 family of putative NAD-dependent histonedeacetylases, which are involved in aging and chromatin structure andsome of which may have NAD-dependent mono-ADP-ribosyltransferaseactivity has moderate similarity to a region of S. cerevisiae Sir2p 267503498CD1 g3249541 8.3E−152 [Homo sapiens] ribonuclease P proteinsubunit p40 Jarrous, N. (1998) RNA 4: 407-417 Autoantigenic propertiesof some protein subunits of catalytically active complexes of humanribonuclease P 364641|RPP40 7.3E−153 [Homo sapiens] [Hydrolase; Nuclease(endo, exo, ribo, deoxyribo)] [Nuclear] Subunit p40 of ribonuclease Pribonucleoprotein, which processes 5′ ends of precursor tRNAs, does notreact with Th sera from patients with systemic sclerosis 27 7504119CD1g21039484 1.0E−132 [fl][Mus musculus] transcription factor b1 g134230979.7E−41 [Caulobacter crescentus] dimethyladenosine transferase475717|LOC51106 5.2E−150 [Homo sapiens] [RNA-binding protein] Member ofthe ribosomal RNA adenine dimethylase family 249581|T03F1.7 1.7E−60[Caenorhabditis elegans] [Transferase] [Nuclear ucleolus; Nuclear]Protein with similarity to ribosomal RNA adenine dimethylases, has weaksimilarity to S. cerevisiae dimethyladenosine transferase Dim1p373375|SPBC336.02 7.0E−12 [Schizosaccharomyces pombe] [Transferase]Dimethylase 28 71532805CD1  g307388 1.7E−74 [Homo sapiens] ribosomalprotein L7 Seshadri, T. et al. (1993) J. Biol. Chem. 268: 18474-18480Identification of a transcript that is down-regulated in senescent humanfibroblasts: Cloning, sequence analysis and regulation of the human L7ribosmal protein gene 337748|RPL7 1.1E−74 [Homo sapiens] [Structuralprotein; Ribosomal subunit; RNA-binding protein] [Cytoplasmic] Ribosomalprotein L7, component of the large 60S ribosomal subunit; expression isreduced in senescent cells 586417|Rpl7 1.7E−74 [Mus musculus][Structural protein; Ribosomal subunit; RNA-binding protein][Cytoplasmic] Ribosomal protein L7, component of the large 60S ribosomalsubunit 246052|F53G12.10 1.1E−72 [Caenorhabditis elegans] [RNA-bindingprotein] [Cytoplasmic] Member of the ribosomal protein L7 protein family370375|rpl7-2 6.9E−71 [Schizosaccharomyces pombe] [Ribosomal subunit]60S ribosomal protein L7B/L7-C 376060|rpl7-1 4.9E−70[Schizosaccharomyces pombe] [Ribosomal subunit] 60S ribosomal proteinL7A 29 5502992CD1 g7230509 0.0 [Drosophila melanogaster] KISMET-L longisoform Therrien, M. et al. (2000) Genetics 156: 1231-1242 A geneticscreen for modifiers of a kinase suppressor of ras-dependent rough eyephenotype in Drosophila 619200|KIAA1564 0.0 [Homo sapiens] Protein ofunknown function, has a region of moderate similarity to a region ofhuman ZFH, which is a zinc finger helicase and a member of the DNAhelicase superfamily II 249667|T04D1.4 1.9E−296 [Caenorhabditis elegans][Helicase] [Nuclear] Member of the DNA helicase protein family691900|FLJ12178 5.5E−149 [Homo sapiens] Protein of unknown function, hasmoderate similarity to a region of human SMARCA2, which is atranscription cofactor that cooperates with glucocorticoid receptor toactivate transcription and is excluded from condensed chromosomes247007|H06O01.2 7.9E−145 [Caenorhabditis elegans] [Helicase] [Nuclear]Putative chromodomain helicase DNA binding protein 30 7503828CD1g1549241 0.0 [Homo sapiens] SWI/SNF complex 170 KDa subunit Wang. W. etal. (1996) Genes Dev. 10: 2117-2130 Diversity and specialization ofmammalian SWI/SNF complexes. 319202|Smarcc1 0.0 [Mus musculus][Transcription factor] [Nuclear] SWI-SNF related matrix associated actindependent regulator of chromatin subfamilyc member 1, chromatin bindingprotein implicated in regulation of transcription by remodelingchromatin, may play role in T cell development and regulation ofapoptosis 338138|SMARCC2 0.0 [Homo sapiens] [Transcription factor][Nuclear] Member 2 of subfamily c of SWI/SNF related matrix associatedactin dependent regulators of chromatin, part of a complex involved infetal to adult globin gene switching and part of a co- repressor complex338136|SMARCC1 0.0 [Homo sapiens] [Transcription factor] [Nuclear]SWI-SNF related matrix associated actin dependent regulator of chromatinsubfamilyc member 1, a putative trancription co-activator which isimplicated in regulation of transcription by remodeling nucleosomes andchromatin 441839|psa-1 5.0E−135 [Caenorhabditis elegans] [DNA-bindingprotein] [Nuclear] Putative component of a SWI/SNF chromatin remodelingcomplex, active in the control of mitosis 372067|SPAC23 2.2E−77[Schizosaccharomyces pombe] Protein with moderate similarity to S.cerevisiae H3.10 Rsc8p 31 2647325CD1 g55471 1.8E−37 [Mus musculus]Zfp-29 Denny, P. and Ashworth, A. (1991) Gene 106: 221-227 A zinc fingerprotein-encoding gene expressed in the post-meiotic phase ofspermatogenesis. 322628|Zfp29 1.6E−38 [Mus musculus] [Transcriptionfactor; DNA-binding protein] Zinc-finger protein that may regulatepost-meiotic germ cell gene expression, expressed specifically inpost-meiotic round spermatids 339004|ZNF84 1.0E−37 [Homo sapiens][Inhibitor or repressor; DNA-binding protein; Transcription factor][Nuclear] Protein containing a KRAB (kruppel-associated box) domainwhich may mediate transcriptional repression and several C2H2 type zincfinger domains, which bind nucleic acids 338982|ZNF205 1.2E−36 [Homosapiens] [Inhibitor or repressor; Transcription factor] Proteincontaining C2H2 type zinc finger domains, which bind nucleic acids, anda KRAB (kruppel- associated box) domain, which may mediatetranscriptional repression 338968|ZNF157 1.2E−36 [Homo sapiens][Inhibitor or repressor; Transcription factor] Zinc finger protein 157,a zinc-finger protein that contains two Kruppel-associated box (KRAB-Aand KRAB-B) transcription repression domains 319698|Zfp46 1.9E−36 [Musmusculus] Zinc finger protein 46, contains an acidic domain followed byC2H2 zinc finger domains in the N-terminal region, may bind to nucleicacids 32 7495416CD1 g488551 1.5E−77 [Homo sapiens] zinc finger proteinZNF132 Tommerup, N. and Vissing, H. (1995) Genomics 27: 259-264Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAsidentify putative candidate genes for developmental and malignantdisorders. 476113|LOC51333 3.2E−160 [Homo sapiens] [DNA-binding protein]Protein containing aC2H2 type zinc finger domain, which bind nucleicacids 423343|KIAA0326 9.5E−81 [Homo sapiens] [DNA-binding protein]Protein containing nineteen C2H2 type zinc finger domains, which bindnucleic acids 338948|ZNF132 1.3E−78 [Homo sapiens] [Transcriptionfactor] Zinc finger protein 132, a member of the Kruppel zinc-fingerprotein family, contains tandemly repeated C2H2 zinc finger domains324156|Mm.10509 3.4E−78 [Mus musculus] [DNA-binding protein] [Nuclear]Protein containing a C2H2 type zinc finger domain, which bind nucleicacids 338954|ZNF135 4.3E−78 [Homo sapiens] Member of the Kruppel familyof zinc-finger proteins 33 8096177CD1 g7243633 5.7E−131 [Homo sapiens]RB-associated KRAB repressor Skapek, S. X. et al. (2000) J. Biol. Chem.275: 7212-7223 Cloning and characterization of a novelKruppel-associated box family transcriptional repressor that interactswith the retinoblastoma gene product, RB 610561|LOC57209 2.5E−201 [Homosapiens] [DNA-binding protein] Protein containing seven C2H2 type zincfinger domains, which bind nucleic acids, has high similarity to aregion of human ZNF33A, which is a zinc finger protein 424090|KIAA09722.5E−137 [Homo sapiens] [Inhibitor or repressor; Transcription factor]Protein containing a KRAB (kruppel-associated box) domain which maymediate protein-protein intereactions, contains C2H2 type zinc fingerdomains, which bind nucleic acids, has moderate similarity totranscriptional repressors 598470|FLJ10469 1.0E−133 [Homo sapiens]Inhibitor or repressor; Transcription factor; DNA-binding protein][Nuclear] Protein containing a KRAB (kruppel-associated box) domainwhich may mediate transcriptional repression, and fourteen C2H2 typezinc finger domains, which bind nucleic acids 437244|RBAK 5.0E−132 [Homosapiens] Inhibitor or repressor; DNA-binding protein; Transcriptionfactor] [Nuclear] RB-associated KRAB protein, a member of the Kruppel-associated box family of transcriptional repressors, interacts with theretinoblastoma protein RB1 and may repress E2F-dependent genes339004|ZNF84 9.6E−129 [Homo sapiens] [Inhibitor or repressor;DNA-binding protein; Transcription factor] [Nuclear] Protein containinga KRAB (kruppel-associated box) domain which may mediate transcriptionalrepression and several C2H2 type zinc finger domains, which bind nucleicacids 34 666763CD1 g12232096 4.2E−18 [Caenorhabditis elegans]replication licensing factor MCM2/3/5-type protein 253521|ZK632.13.7E−19 [Caenorhabditis elegans] [DNA-binding protein] [Nuclear] Memberof the MCM initiator complex (DNA replication) protein family 1280|CDC548.8E−17 [Saccharomyces cerevisiae] [Hydrolase; DNA-binding protein;ATPase] [Nuclear] Protein involved in DNA synthesis initiation, memberof the MCM family of DNA-dependent ATPases required for initiation ofDNA replication 637674|orf6.3958 1.1E−16 [Candida albicans] [Hydrolase;DNA-binding protein; ATPase]Protein with high similarity to S.cerevisiae Cdc54p, which is involved in DNA synthesis initiation, memberof the MCM family of DNA-dependent ATPases, which may act as replicativeDNA helicases 35 7504091CD1 g13097225 1.1E−175 [Homo sapiens]mitochondrial ribosomal protein L3 428458|MRPL3 9.4E−177 [Homo sapiens][Structural protein; RNA-binding protein; Ribosomal subunit] [Nuclear;Nuclear nucleolus; Cytoplasmic] Ribosomal protein L3, component of thelarge 60S ribosomal subunit, may be involved in binding of the mRNA tothe ribosome 713842|C26E6.6 5.8E−39 [Caenorhabditis elegans] Proteinwith weak similarity to ribosomal protein L3 7135|MRPL9 7.0E−33[Saccharomyces cerevisiae] [RNA-binding protein; Ribosomal subunit][Mitochondrial] Mitochondrial ribosomal protein of the large subunit(YmL9; E. coli L3; human MRL3) 647232|orf6.8737 4.2E−31 [Candidaalbicans] [RNA-binding protein; Ribosomal subunit][Cytoplasmic] Proteinwith high similarity to S. cerevisiae Mrp19p, which is a mitochondrialribosomal protein of the large subunit, protein of the large 60Sribosomal subunit 616118|SPAC644.17c 4.1E−29 [Schizosaccharomyces pombe]Mitochondrial ribosomal protein L9 36 7503568CD1 g13172240 1.2E−161 [Musmusculus] alpha-CP2; hnRNP-E2 Makeyev, AV, Liebhaber, SA. Genomics(2000) Genomics 67: 301-316 Identification of two novel mammalian genesestablishes a subfamily of KH- domain RNA-binding proteins. 743096|PCBP24.1E−161 [Homo sapiens] [RNA-binding protein] Protein containingKHRNA-binding domains, a major poly(rC)-binding protein togetherwithPCBP1 and HNRPK 343616|PCBP1 1.7E−138 [Homo sapiens] [RNA-bindingprotein] Poly(rC)-binding protein 1, contains KH RNA-binding domains,binds poly(rC) RNA, acts as a translational repressor and plays a rolein mRNA stability 430118|Pcbp1 1.7E−138 [Mus musculus] [RNA-bindingprotein] Poly(rC)-binding protein 1, contains KH RNA-binding domains,binds poly(rC) RNA and may play a role in mRNA stability 613185|PCBP34.3E−129 [Homo sapiens] [RNA-binding protein] Poly(rC)-binding protein3, a member of a family of KH-domain containing RNA-binding proteins618966|Pcbp3 3.8E−128 [Mus musculus] [Nuclear] Protein with highsimilarity to murine Pcbp2 (secreted phosphoprotein), which containsKHRNA-binding domains and binds preferentially to oligo dC 37 7504101CD1g882258 0.0 [Homo sapiens] chromatin assembly factor-I p150 subunitKaufman, P.D. et al. (1995) Cell 81: 1105-1114 The p150 and p60 subunitsof chromatin assembly factor I: a molecular link between newlysynthesized histones and DNA replication. 341970|CHAF1A 0.0 [Homosapiens] [Complex assembly protein; Chaperones; DNA-binding protein][Nuclear] Chromatin assembly factor 1 subunit A, chromatin assemblyfactor 1 subunit that mediates deposition of newly synthesized histonesH3 and acetylated H4 onto replicated DNA, may mediate a chromatinassembly response to DNA damage by interacting with PCNA 433052|Chaf1a2.0E−224 [Mus musculus] [Complex assembly protein; DNA-binding protein][Nuclear] Chromatin assembly factor 1 subunit A, chromatin assemblyfactor 1 subunit that interacts with HP1 proteins, may modulatechromatin and heterochromatin dynamics; human CHAF1A may mediate achromatin assembly response to DNA damage by interacting with PCNA441141|T06D10.2 9.5E−35 [Caenorhabditis elegans] Protein with moderatesimilarity to C. elegans F36H12.3 631024|orf6.633 3.2E−26 [Candidaalbicans] Protein of unknown function, has low similarity to S.cerevisiae Rlf2p, which is a subunit of the chromatin assembly complexinvolved in nucleosome assembly linked with DNA replication639148|orf6.4695 3.2E−26 [Candida albicans] Protein of unknown function,has low similarity to S. cerevisiae Rlf2p, which is subunit 1 of thechromatin assembly complex involved in nucleosome assembly linked withDNA replication 38 6946680CD1 g13560888 8.6E−160 [Homo sapiens]EZFIT-related protein 1 308339|ZNF184 1.7E−154 [Homo sapiens]Kruppel-like zinc-finger protein, maximally expressed in testis,moderately in other tissues 339006|ZNF85 1.6E−142 [Homo sapiens][Inhibitor or repressor; Transcription factor; DNA-binding protein][Nuclear] Zinc finger protein 85, member of the ZNF91 family ofKruppel-associated box (KRAB) zinc finger proteins, functions as atranscriptional co-repressor 432896|ZNF208 2.7E−140 [Homo sapiens] Zincfinger protein 208, a ubiquitously expressed Kruppel- associated box(KRAB) zinc finger protein 339004|ZNF84 4.4E−140 [Homo sapiens][Inhibitor or repressor; DNA-binding protein; Transcription factor][Nuclear] Protein containing a KRAB (kruppel-associated box) domainwhich may mediate transcriptional repression and several C2H2 type zincfinger domains, which bind nucleic acids 475040|HSPC059 3.6E−138 [Homosapiens] [Inhibitor or repressor; Transcription factor; DNA-bindingprotein] Protein containing sixteen C2H2 type zinc finger domains, whichbind nucleic acids, contains a KRAB (kruppel-associated box) domainwhich may mediate transcriptional repression 39 7001142CD1 g135608887.8E−166 [Homo sapiens] EZFIT-related protein 1 308339|ZNF184 2.2E−145[Homo sapiens] Kruppel-like zinc-finger protein, maximally expressed intestis, moderately in other tissues 619192|KIAA1559 2.4E−141 [Homosapiens] Protein with strong similarity to murine Zfp30, which is azinc- finger protein containing a Kruppel-associated box (KRAB)transcriptional repression domain 424068|KIAA0961 1.5E−139 [Homosapiens] Protein with strong similarity to murine Zfp30, which is azinc- finger protein containing a Kruppel-associated box (KRAB)transcriptional repression domain 475040|HSPC059 6.7E−135 [Homo sapiens][Inhibitor or repressor; Transcription factor; DNA-binding protein]Protein containing sixteen C2H2 type zinc finger domains, which bindnucleic acids, contains a KRAB (kruppel-associated box) domain which maymediate transcriptional repression 339004|ZNF84 8.5E−135 [Homo sapiens][Inhibitor or repressor; DNA-binding protein; Transcription factor][Nuclear] Protein containing a KRAB (kruppel-associated box) domainwhich may mediate transcriptional repression and several C2H2 type zincfinger domains, which bind nucleic acids 40 71158380CD1 g45192702.8E−264 [Homo sapiens] Kruppel-type zinc finger protein Katoh, O. etal. (1998) Biochem. Biophys. Res. Commun. 249: 595-600 ZK1, a novelKruppel-type zinc finger gene, is induced following exposure to ionizingradiation and enhances apoptotic cell death on hematopoietic cells700794|FLJ14356 1.6E−284 [Homo sapiens] Protein with high similarity tohuman ZNF136, which is a C2H2 zinc-finger protein that repressestranscription when fused to the heterologous KRAB B subdomain of humanZNF10 342918|ZK1 2.5E−265 [Homo sapiens] Kruppel-type zinc fingerprotein, has an A box of Kruppel- associated box (KRAB) domain andfifteen zinc finger motifs, possibly functions in radiation-inducedapoptosis, expression is induced by exposure to ionizing radiation476341|GIOT-2 2.6E−254 [Homo sapiens] [Inhibitor or repressor;Transcription factor; DNA-binding protein] Protein containing fifteenC2H2 type zinc finger domains, which bind nucleic acids, also contains aKRAB (kruppel-associated box) domain which may mediate transcriptionalrepression 594469|HSZFP36 3.6E−248 [Homo sapiens] [Inhibitor orrepressor; Transcription factor; DNA-binding protein] Protein containingfourteen C2H2 type zinc finger domains, which bind nucleic acids, alsocontains a KRAB (kruppel-associated box) domain which may mediatetranscriptional repression 476345|LOC51712 1.5E−203 [Homo sapiens][Inhibitor or repressor; Transcription factor; DNA-binding protein]Protein containing eighteen C2H2 type zinc finger domains, which bindnucleic acids, also contains a KRAB (kruppel-associated box) domainwhich may mediate transcriptional repression 41 7503861CD1 g147644991.1E−171 [Homo sapiens] zinc finger protein 346716|KIAA0211 0.0 [Homosapiens] Protein containing C2H2 type zinc finger domains, which bindnucleic acids 598616|FLJ10697 1.1E−111 [Homo sapiens] [DNA-bindingprotein] Protein with a low similarity to KRAB zinc finger proteins423343|KIAA0326 4.7E−21 [Homo sapiens] [DNA-binding protein] Proteincontaining nineteen C2H2 type zinc finger domains, which bind nucleicacids 342394|ZNF256 3.4E−20 [Homo sapiens] [Inhibitor or repressor;Transcription factor; DNA-binding protein] Zinc finger protein 256, aputative transcriptional repressor that may play a role in hemopoiesis,member of the Kruppel-like zinc-finger family Han, Z. G. et al. (1999)J. Biol. Chem. 274: 35741-35748 Molecular cloning of six novelKruppel-like zinc finger genes from hematopoietic cells andidentification of a novel transregulatory domain KRNB. 338994|ZNF431.7E−19 [Homo sapiens] Zinc finger protein 43, contains C2H2 zinc fingermotifs, expressed mainly in B and T cells 42 7758395CD1 g155531399.6E−104 [Homo sapiens] (AF297872) zinc finger transcription factorTReP-132 Gizard, F. (2001) J. Biol. Chem. 276: 33881-33892 A novel zincfinger protein TReP-132 interacts with CBP/p300 to regulate humanCYPI1A1 [steroid synthesis] gene expression 594951|HSA277276 4.2E−99[Homo sapiens] [DNA-binding protein] [Nuclear] Protein containing aMyb-like DNA-binding domain and two C2H2 type zinc finger domain, whichbind nucleic acids 246070|F53H10.2 3.3E−22 [Caenorhabditis elegans]Protein with weak similarity to C. elegans D1014.9 gene product614095|Brd4 3.6E−12 [Mus musculus] Mitotic chromosome-associatedprotein, a member of the bromodomain superfamily BET subgroup,associates with mitotic chromosomes and functions in chromosomaldynamics during G(2)/M transition 645094|orf6.7668 2.8E−11 [Candidaalbicans] Protein containing a pleckstrin homology (PH) domain, whichmediate protein-protein and protein-lipid interactions, has a region oflow similarity to a region of S. pombe Php5p, which is a subunit ofCCAAT-binding factor 43 71039312CD1 g7296687 1.4E−65 [Drosophilamelanogaster] cas gene product Adams, M. D. et al. (2000) The genomesequence of Drosophila melanogaster. Science 287: 2185-2195.599292|FLJ20321 0.0 [Homo sapiens] [DNA-binding protein] [Nuclear]Protein containing five C2H2 type zinc finger domains, which bindnucleic acids. 44 7291318CD1 g5640019 4.0E−52 [Mus musculus] zinc fingerprotein ZFP235 658380|ZFP93 1.3E−54 [Homo sapiens] Member of theXRCC1-linked KRAB zinc-finger protein family, has similarity tomurineZfp93. Shannon, M. et al. (1996) Comparative analysis of a conservedzinc finger gene cluster on human chromosome 19q and mouse chromosome 7.Genomics 33: 112-20. 570912|ZNF226 4.0E−54 [Homo sapiens] [Inhibitor orrepressor; Transcription factor; DNA-binding protein] Protein containingeighteen C2H2 type zinc finger domains, which bind nucleic acids, a KRAB(kruppel-associated box) domain which may mediate transcriptionalrepression. 434624|ZNF234 2.1E−52 [Homo sapiens] Member of theKruppel-related zinc finger protein family. Abrink, M. et al. (2001)Conserved interaction between distinct Kruppel- associated box domainsand the transcriptional intermediary factor 1 beta. Proc. Natl. Acad.Sci. U.S.A. 98: 1422-1426. 45 2638619CD1 g2529737 5.7E−76 [Xenopuslaevis] ER1 Paterno, G.D. et al. (1997) cDNA cloning of a novel,developmentally-regulated immediate early gene activated by fibroblastgrowth factor and encoding a nuclear protein. J. Biol. Chem. 272:25591-25595. 556774|KIAA1193 1.1E−296 [Homo sapiens] [DNA-bindingprotein] Protein containing a Myb DNA-binding domain, and anuncharacterized ELM2 domain, which are found in C. elegans egl- 27 andhuman and rat MTA1. 46 2810014CD1 g6601438 9.6E−35 [Homo sapiens] AF5q31protein Taki, T. et al. (1999) AF5q31, a newly identified AF4-relatedgene, is fused to MLL in infant acute lymphoblastic leukemia with ins(5;11)(q31; q13q23) Proc. Natl. Acad. Sci. U.S.A. 96: 14535-14540.436208|AF5Q31 8.4E−36 [Homo sapiens] [DNA-binding protein; Transcriptionfactor] ALL1 fused gene from 5q31, a putative transcription factor;corresponding gene is fused to MLL in cases of acute lymphoblasticleukemia as a result of genetic rearrangements. Taki, T. et al. (1999)AF5q31, a newly identified AF4-related gene, is fused to MLL in infantacute lymphoblastic leukemia with ins (5; 11) (q31; q13q23). Proc. Natl.Acad. Sci. U.S.A. 96: 14535-14540. Hillman, M. A. and Gecz, J. (20001)Fragile XE-associated familial mental retardation protein 2 (FMR2) actsas a potent transcription activator. J. Hum Genet. 46: 251-259. 473457155CD1 g5811583 0.0 [Rattus norvegicus] TIP120-family proteinTIP120B Aoki, T. et al. (1999) TIP120B: a novel TIP120-family proteinthat is expressed specifically in muscle tissues. Biochem. Biophys. Res.Commun. 261: 911-916. 423639|KIAA0667 0.0 [Homo sapiens] TBP-interactingprotein 120B. 600204|TIP120 0.0 [Homo sapiens] [Nuclear] mRNA forKIAA0829 gene, isolated from human brain cDNA library. 332994|Rn.329340.0 [Rattus norvegicus] [Transcription factor] [Nuclear] TBP-interactingprotein that may play a role in transcriptional regulation. 487435171CD1 g3395529 8.7E−183 [Mus musculus] homeodomain protein583221|Hmx3 6.6E−146 [Mus musculus] [Transcription factor; DNA-bindingprotein] H6 homeobox 3, a DNA binding protein that is required for theformation of the inner ear vestibular system, may function in neuronalcell specification; deficiency causes reproductive defects in femalesand balance defects. Wang, W. et al. (1998) Inner ear and maternalreproductive defects in mice lacking the Hmx3 homeobox gene. Dev. Suppl.125: 621-634. 49 7499936CD1 g9931482 2.2E−81 [Cloning vector pFB-ERV]retinoic acid receptor RXR 321064|Rxra 3.5E−85 [Mus musculus][Activator; Transcription factor; DNA-binding protein; Receptor(signalling)] [Nuclear] Retinoid X receptor alpha, a high affinityreceptor for 9-cis retinoic acid, controls multiple metabolic pathwaysby interacting with a variety of nuclear receptors and regulatingtranscriptional activity. Mangelsdorf, D. J. et al. (1990) Nuclearreceptor that identifies a novel retinoic acid response pathway. Nature345: 224-229. 717364|1fm6_A 2.0E−82 [Protein Data Bank] Retinoic AcidReceptor Rxr-Alpha. Fournes, B. et at. (2001) The CEACAM1-L Ser503residue is crucial for inhibition of colon cancer cell tumorigenicity.Oncogene 20: 219-230. 50 7504125CD1 g531523 1.1E−71 [Homo sapiens] NetGiovane, A. et al. (1994) Net, a new ets transcription factor that isactivated by Ras Genes Dev. 8: 1502-1513. 342016|ELK3 9.7E−73 [Homosapiens] [DNA-binding protein; Transcription factor] [Nuclear] ETS-domain protein (SRF accessory protein 2), a member of the ets family oftranscription factors that regulates transcription and serves as atarget of Ras- MAPK signal transduction pathways. Price, M. A. (1995)Comparative analysis of the ternary complex factors Elk-1, SAP-1 a andSAP-2 (ERP/NET). Embo Journal 14: 2589-2601. 51 7505742CD1 g5163813.5E−266 [Homo sapiens] transcription factor Murphy, D.B. et al. (1994)Human brain factor 1, a new member of the fork head gene family.Genomics 21: 551-557. 342038|FOXG1B 3.0E−267 [Homo sapiens] [DNA-bindingprotein; Transcription factor] Member of the HNF- 3/fork head family oftranscriptional regulators, expression is limited to the neuronal cellsin the telencephalon. Pierrou, S. et al. (1994) Cloning andcharacterization of seven human forkhead proteins: binding sitespecificity and DNA bending. Embo Journal 13: 5002-5012. 52 7505757CD1g5811585 0.0 [Rattus norvegicus] TIP120-family protein TIP120B,alternatiely spliced form Aoki, T. et al. (1999) TIP120B: a novelTIP120-family protein that is expressed specifically in muscle tissues.Biochem. Biophys. Res. Commun. 261: 911-916. 423639|KIAA0667 0.0 [Homosapiens] TBP-interacting protein 120B. 600204|TIP120 0.0 [Homo sapiens][Nuclear] mRNA for KIAA0829 gene, isolated from human brain cDNAlibrary. Yogosawa, S. et al. (1999) Induced expression, localization,and chromosome mapping of a gene for the TBP-interacting protein 120A.Biochem. Biophys. Res. Commun. 266: 123-128. 53 7504126CD1 g37179781.4E−41 [Mus musculus] 5S ribosomal protein Vizirianakis, I.S. et al.(1999) Expression of ribosomal protein S5 cloned gene duringdifferentiation and apoptosis in murine erythroleukemia (MEL) cells.Oncol. Res. 11: 409-419. 709567|RPS5 1.4E−43 [Homo sapiens] [Structuralprotein; RNA-binding protein; Ribosomal subunit] [Cytoplasmic] Ribosomalprotein S5, a component of the 40S ribosomal subunit; gene expression isaltered in colorectal carcinoma cells. Vizirianakis, I. S. et al. (1999)Expression of ribosomal protein S5 cloned gene during differentiationand apoptosis in murine erythroleukemia (MEL) cells. Oncol. Res. 11:409-419. 54 7504099CD1 g871299 2.9E−173 [Homo sapiens] Human pre-mRNAcleavage factor I 68 kDa subunit Ruegsegger, U. et al. (1998) Humanpre-mRNA cleavage factor Im is related to spliceosomal SR proteins andcan be reconstituted in vitro from recombinant subunits. Mol. Cell 1:243-253. 428272|CPSF6 2.5E−174 [Homo sapiens] [RNA-binding protein][Nuclear] Cleavage and polyadenylation specific factor 6, a putativemRNA-binding protein that is the 68 kDa subunit of the mRNA cleavagefactor Im (CF Im) complex, plays a role in pre-mRNA 3′ end processing.de Vries, H. et al. (2000) Human pre-mRNA cleavage factor II(m) containshomologs of yeast proteins and bridges two other cleavage factors EmboJournal 19: 5895-5904. 55 7505733CD1 g2098734 1.6E−39 [Homo sapiens]integrase 476591|HSU88895 1.2E−52 [Homo sapiens] Putative proteinencoded by human endogenous retrovirus H (HERV-H). Lindeskog, M. andBlomberg, J. (1997) Spliced human endogenous retroviral HERV-H envtranscripts in T-cell leukaemia cell lines and normal leukocytes:alternative splicing pattern of HERV-H transcripts [published erratumappears in J. Gen. Virol. 1998 Jan; 79 (Pt 1): 212] J. Gen. Virol.,2575-2585. 56 7959829CD1 g9652099 1.0E−69 [Mus musculus] pseudouridinesynthase 3 Chen, J. and Patton, J. R. (2000) Pseudouridine synthase 3from mouse modifies the anticodon loop of tRNA. Biochemistry 39:12723-12730. 703953|FKSG32 9.1E−85 [Homo sapiens] Protein with moderatesimilarity to S. cerevisiae Deg1p, which is a pseudouridine synthasethat catalyzes the formation of pseudouridine-38 and -39 in cytoplasmicand mitochondrial tRNAs. 57 7502168CD1 g52977 3.9E−212 [Mus musculus]modifier 3 (M33) Pearce, J. J. et al. (1992) The mouse has aPolycomb-like chromobox gene. Development 114: 921-929. 321346|Cbx23.4E−213 [Mus musculus] Homolog of Drosophila polycomb chromobox, whichis implicated in clonal inheritance of determined states through effectson chromatin structure; mutation in the gene causes sex reversal.Katoh-Fukui, Y. et al. (1998) Male-to-female sex reversal in M33 mutantmice. Nature 393: 688-692. 58 7503888CD1 g10946128 0.0 [Homo sapiens]SMARCA4 isoform 1 Wong, A. K. C. et al. (2000) BRG1, a component of theSWI-SNF complex, is mutated in multiple human tumor cell lines. CancerRes. 60: 6171-6177. 338130|SMARC 0.0 [Homo sapiens] [Hydrolase;Activator; Helicase; Transcription factor; ATPase] A4 [Nuclear] SWI-SNFrelated matrix associated actin-dependent regulator of chromatinsubfamily, a member 4, mediates transcriptional regulation by nuclearreceptors, RB1, Myc, KLF1 and BRCA1, involved in cell cycle control andT cell receptor signaling. Kadam, S. et al. (2000) Functionalselectivity of recombinant mammalian SWI/SNF subunits Genes AndDevelopment 14: 2441-2451.

TABLE 3 Amino Potential Analytical SEQ Incyte Acid Potential Glycosyl-Methods ID Polypep- Resi- Phosphorylation ation and NO: tide ID duesSites Sites Signature Sequences, Domains and Motifs Databases 17503848CD1 1374 S83 S87 S88 S120 N109 N166 PROTEIN TREACHER COLLINSSYNDROME BLAST_(—) S153 S156 S171 N572 N759 TREACLE PUTATIVE NUCLEOLARPRODOM S198 S199 S205 N1180 TRAFFICKING PHOSPHOPROTEIN REPEAT S206 S217S264 PD017611: P1048-P1322 S265 S270 S272 S288 S304 S335 S336 S340 S342S353 S369 S400 S401 S405 S407 S470 S471 S479 S533 S576 S617 S618 S622S624 S687 S688 S692 S707 S724 TREACHER COLLINS SYNDROME PROTEINBLAST_(—) S793 S794 S798 TREACLE DISEASE MUTATION PRODOM S800 S813 S829POLYMORPHISM PUTATIVE NUCLEOLAR S857 S858 S862 PD017236: Q563-R911 S864S920 S922 S924 S965 S969 S1027 S1039 S1041 S1063 S1076 S1077 S1143 S1160S1223 S1224 S1230 S1236 S1247 S1324 S1331 S1351 S1359 T45 T98 T102TREACHER COLLINS SYNDROME TREACLE BLAST_(—) T129 T144 T173 PROTEINDISEASE MUTATION PRODOM T210 T609 T785 POLYMORPHISM PD038028: A411-P562T906 T916 T983 T1007 T1067 T1072 T1108 T1219 T1244 T1271 T1369 PROTEINTREACHER COLLINS SYNDROME BLAST_(—) TREACLE PUTATIVE NUCLEOLAR PRODOMTRAFFICKING PHOSPHOPROTEIN REPEAT PD016387: P103-A250 ACIDIC SERINECLUSTER REPEAT DM04746 BLAST_DOMO |S57757|1-646: E9-T629 |P41777|1-386:K502-E828 |I38073|1-377: M1-S369 do NEUROFILAMENT; TRIPLET; BLAST_DOMODM04498|P12036|434-1019: T210-S798 Atp_Gtp_A: A149-S156, A310-T317,A663-T670, MOTIFS A835-T842 2 2608080CD1 588 S103 S112 S151 N302 N358signal_cleavage: M1-T17 SPSCAN S209 T19 T41 T69 N379 N470 T173 T200 T293T334 T349 T405 KRAB box: V9-K71 HMMER_PFAM Zinc finger, C2H2 type:Y227-H249, F367-H389, HMMER_PFAM L479-H501, F423-H445, Y395-H417,Y507-H529, Y199-H221, F535-H557, F563-H585, Y283-H305, Y451-H473,Y339-H361, Y255-H277, F311-H333 Zinc finger, C2H2 type, domain proteinsBL00028: BLIMPS_(—) C257-H273 BLOCKS C2H2-type zinc finger signaturePR00048: P254- BLIMPS_(—) R267, L550-G559 PRINTS PROTEIN ZINC-FINGERMETAL PD00066: H245- BLIMPS_(—) C257 PRODOM PROTEIN ZINC FINGER ZINCPD01066: F111-G49 BLIMPS_(—) PRODOM PROTEIN ZINC FINGER METAL BINDINGDNA BLAST_(—) BINDING ZINC FINGER PATERNALLY PRODOM EXPRESSED ZN FINGERPW1 PD017719: G251- H501; G223-V465; P310-H557 HYPOTHETICAL ZINC FINGERPROTEIN BLAST_(—) B03B8.4 IN CHROMOSOME III ZINC FINGER PRODOM DNABINDING METAL BINDING NUCLEAR PD149420: R307-G475 ZINC FINGER DNABINDING PROTEIN METAL BLAST_(—) BINDING NUCLEAR ZINC FINGER PRODOMTRANSCRIPTION REGULATION REPEAT PD000072: K393-C456 ZINCFINGER METALBINDING DNA BINDING BLAST_(—) PROTEIN FINGER ZINC NUCLEAR REPEAT PRODOMTRANSCRIPTION REGULATION PD001562: V9- K71 KRAB BOX DOMAIN DM00605BLAST_DOMO |I48689|11-85: V9-C79 |P51786|24-86: V9-W68 |P52736|1-72:V9-C79 ZINC FINGER, C2H2 TYPE, DOMAIN BLAST_DOMO DM00002|Q05481|789-829:R247-E287; R387-E427; K414-E455 Zinc_Finger_C2h2: C201-H221, C229-H249,C257- MOTIFS H277, C285-H305, C313-H333, C341-H361, C369- H389,C397-H417, C425-H445, C453-H473, C481- H501, C509-H529, C537-H557,C565-H585 3 7503402CD1 607 S65 S69 S151 S276 N272 N410 signal_cleavage:M1-A39 SPSCAN S481 S586 T35 T91 N479 N573 T195 T279 T368 T394 T443 Zincfinger, C2H2 type: Y266-H290, Y236-H260, HMMER_PFAM F206-H230,Y386-H409, Y326-H350, Y356-H380, F296-H320 Zinc finger, C2H2 type,domain proteins BL00028: BLIMPS_(—) C388-H404 BLOCKS PROTEIN ZINC-FINGERMETA PD00066: H256- BLIMPS_(—) C268 PRODOM SELENOCYSTEINE TRNA PROTEINBLAST_(—) TRANSCRIPTION ACTIVATING FACTOR DNA PRODOM BINDING ZINC FINGERMETAL BINDING ZINC PD016532: D38-G172 SELENOCYSTEINE TRNA TRANSCRIPTIONBLAST_(—) ACTIVATING FACTOR PROTEIN DNA BINDING PRODOM ZINC FINGER METALBINDING GENE PD016467: H382-D449 TRANSCRIPTION SELENOCYSTEINE TRNABLAST_(—) PROTEIN ACTIVATING FACTOR GENE ZINC PRODOM FINGER REGULATIONPD155356: V450-S518 SELENOCYSTEINE TRNA PROTEIN BLAST_(—) TRANSCRIPTIONACTIVATING FACTOR DNA PRODOM BINDING ZINC FINGER METAL BINDING ZINCPD034459: V519-G600 do ACTIVATING; SELENOCYSTEINE; TRNA; BLAST_DOMODM04750 |P52747|427-625: A408-D607 |S58681|465-600: G489-D606 ZINCFINGER, C2H2 TYPE, DOMAIN DM00002 BLAST_DOMO |P52747|212-244: R193-H226|P52747|396-425: H377-T407 Zinc_Finger_C2h2: C208-H230, C238-H260, C268-MOTIFS H290, C298-H320, C328-H350, C358-H380, C388- H409 4 7503517CD1422 S89 S193 S339 T55 ADP-glucose pyrophosphorylase proteins BL00808:BLIMPS_(—) T163 T367 A5-P24, V101-K134, G335-V366 BLOCKS TRANSLATIONINITIATION FACTOR EIF2B BLAST_(—) GAMMA SUBUNIT GDPGTP EXCHANGE PRODOMAMINO ACID BIOSYNTHESIS REGULATION PD105480: S212-E30 TRANSLATIONINITIATION FACTOR EIF2B BLAST_(—) GAMMA SUBUNIT GDPGTP EXCHANGE PRODOMAMINO ACID BIOSYNTHESIS PD022735: P141- K189; K189-S211 Rgd: R256-D258MOTIFS 5 7500014CD1 142 S2 S12 S35 S60 N130 S107 S116 T48 6 7501365CD1433 S24 S51 S55 S195 N79 N361 signal_cleavage: M1-A18 SPSCAN S222 S223S379 T178 T197 Signal Peptide: M1-A18; M1-Y20; M1-S24 HMMER PROTEIN CASPCARTILAGE ASSOCIATED BLAST_(—) PRECURSOR SIGNAL NUCLEOLAR PRODOMAUTOANTIGEN NO55 NUCLEAR ANTIGEN PD023886: G17-E276 CASP CARTILAGEASSOCIATED PROTEIN BLAST_(—) PRECURSOR SIGNAL PD155949: L279-R337 PRODOM7 7503540CD1 1450 S51 S122 S126 N64 N495 FHA domain: I23-G90 HMMER_PFAMS135 S239 S309 N516 N618 S313 S356 S379 N670 N814 S391 S482 S538 N1045S559 S634 S667 S699 S709 S731 S788 S835 S860 S903 S914 S944 S950 S961Zinc finger, C2H2 type, domain proteins BL00028: BLIMPS_(—) S968 S988S1014 H387-H403 BLOCKS S1047 S1064 S1067 S1079 S1096 S1100 S1146 S1212S1273 S1291 S1335 S1426 T80 T258 T293 T350 T377 T395 T416 T497 T526 T546T566 T611 T614 T636 T675 T682 T698 T722 T850 T855 T925 T960 T1036 T1041T1131 T1216 T1267 T1271 Y78 Y1157 8 7504326CD1 647 S31 S37 S96 S131 N103N377 WW domain: S131-P160 HMMER_PFAM S142 S155 S202 N415 N529 S308 S379S446 N533 N536 S452 S511 S520 N539 N546 S548 T83 T224 T303 T309 T620 Y25Y129 Y232 WW domain signature PR00403: Y146-P160, S131- BLIMPS_(—) K144PRINTS do MUCIN; MUC5; TRACHEOBRONCHIAL; BLAST_DOMODM05454|S55316|1-317: P249-T541 Rgd: R19-D21 MOTIFS Ww_Domain_1:W135-P160 MOTIFS 9 7504388CD1 195 S64 S111 S154 T22 Rgd: R23-D25 MOTIFST41 10 2828380CD1 781 S17 S49 S127 S142 N118 N164 Zinc finger, C2H2type: Y353-H375, C409-H431, HMMER_PFAM S254 S307 S338 N339 N675Y549-H571, Y493-H515, Y437-H459, Y297-H319, S391 S395 S419 N777Y577-H599, Y465-H487, Y213-H235, Y633-H655, S423 S531 S643 Y717-H739,Y745-H767, Y661-H683, F325-H347, S674 S740 T8 T120 Y605-H627, Y521-H543,Y381-H403, Y185-H207, T166 T209 T333 Y241-H263, Y269-H291, Y689-H711T436 T669 T773 Y161 KRAB box: L7-E67 HMMER_PFAM Zinc finger, C2H2 type,domain proteins BL00028: BLIMPS_(—) C327-H343 BLOCKS PROTEIN ZINC-FINGERMETA PD00066: H595- BLIMPS_(—) C607 PRODOM PROTEIN ZINC FINGER ZINCPD01066: F9-G47 BLIMPS_(—) PRODOM PROTEIN ZINC FINGER METAL BINDING DNABLAST_(—) BINDING ZINC FINGER PATERNALLY PRODOM EXPRESSED ZNFINGER PW1PD017719: G461- H711 HYPOTHETICAL ZINC FINGER PROTEIN BLAST_(—) B03B8.4IN CHROMOSOME III ZINC FINGER PRODOM DNA BINDING METAL BINDING NUCLEARPD149420: E462-H735, I374-H651 MYELOBLAST KIAA0211 ZINC FINGER METALBLAST_(—) BINDING DNA BINDING PD149061: K494-H679 PRODOM ZINC FINGER DNABINDING PROTEIN METAL BLAST_(—) BINDING NUCLEAR ZINC FINGER PRODOMTRANSCRIPTION REGULATION REPEAT PD000072: K519-C582 ZINC FINGER, C2H2TYPE, DOMAIN DM00002 BLAST_DOMO |P52743|31-93: L368-H431|Q05481|789-829: R596-Q637, Q484-E525, R512- E553 |Q05481|831-885:C358-E413 KRAB BOX DOMAIN DM00605|Q03923|1-75: G5- BLAST_DOMO S49 Zincfinger, C2H2 type, domain: C187-H207, C215- MOTIFS H235, C243-H263,C271-H291, C299-H319, C327- H347, C355-H375, C383-H403, C409-H431, C411-H431, C439-H459, C467-H487, C495-H515, C523- H543, C551-H571, C579-H599,C607-H627, C635- H655, C663-H683, C691-H711, C719-H739, C747- H767 116456919CD1 595 S24 S65 S100 S124 N12 N39 Zinc finger, C2H2 type:N172-H194, Y284-H306, HMMER_PFAM S158 S186 S267 N118 N122 Y200-H222,Y256-H278, Y396-H418, Y368-H390, S270 S307 S382 N516 Y424-H448,Y340-H362, Y312-H334, Y458-H480, T14 T36 T242 C228-H250 T537 Y139 KRABbox: V4-Q50 HMMER_PFAM Zinc finger, C2H2 type, domain proteins BL00028:BLIMPS_(—) C398-H414 BLOCKS C2H2-type zinc finger signature PR00048:P367- BLIMPS_(—) S380, L383-G392 PRINTS PROTEIN ZINC-FINGER METAPD00066: H386- BLIMPS_(—) C398 PRODOM PROTEIN ZINC FINGER ZINC PD01066:F6-G44 BLIMPS_(—) PRODOM PROTEIN ZINC FINGER METAL BINDING DNA BLAST_(—)BINDING ZINC FINGER PATERNALLY PRODOM EXPRESSED ZNFINGER PW1 PD017719:G224- K506 ZINC FINGER DNA BINDING PROTEIN METAL BLAST_(—) BINDINGNUCLEAR ZINC FINGER PRODOM TRANSCRIPTION REGULATION REPEAT PD000072:K366-C429 R30385_2 ZINC FINGER PROTEIN BLAST_(—) TRANSCRIPTIONREGULATION DNA BINDING PRODOM REPRESSOR ZINC FINGER METAL BINDINGPD030014: K75-E138 KRAB BOX DOMAIN DM00605|P52737|1-76: M1- BLAST_DOMOD76 KRAB BOX DOMAIN DM00605|I49636|10-85: V4- BLAST_DOMO L66 ZINCFINGER, C2H2 TYPE, DOMAIN DM00002 BLAST_DOMO |Q05481|789-829: E359-Q400|Q05481|831-885: C373-E428 Cell attachment sequence: R126-D128 MOTIFSATP/GTP-binding site motif A (P-loop): A173-T180 MOTIFS Zinc finger,C2H2 type, domain: C174-H194, C202- MOTIFS H222, C228-H250, C230-H250,C286-H306, C314- H334, C342-H362, C370-H390, C398-H418, C426- H448,C460-H480 12 7502244CD1 226 S143 S192 T59 BED zinc finger: S37-R89HMMER_PFAM PHOSPHATE AMINOTRANSFERA PD00040: R49- BLIMPS_(—) H56 PRODOM13 7498718CD1 548 S23 S123 S189 N16 N121 KRAB box: V22-E84 HMMER_PFAMS294 S321 S405 N150 N247 S433 S545 T32 T66 N462 T186 T347 T355 T515 Y311Zinc finger, C2H2 type: Y451-H473, Y395-H417, HMMER_PFAM Y423-H445,Y311-H333, Y507-H529, Y479-H501, Y339-H361, Y367-H389, F209-H231 Zincfinger, C2H2 type, domain proteins BL00028: BLIMPS_(—) C397-H413 BLOCKSC2H2-type zinc finger signature PR00048: P394- BLIMPS_(—) K407,L438-G447 PRINTS PROTEIN ZINC-FINGER META PD00066: H441- BLIMPS_(—) C453PRODOM PROTEIN ZINC FINGER ZINC PD01066: F24-G62 BLIMPS_(—) PRODOMPROTEIN ZINC FINGER METAL BINDING DNA BLAST_(—) BINDING ZINC FINGERPATERNALLY PRODOM EXPRESSED ZNFINGER PW1 PD017719: P310- Y544 ZINCFINGER METAL BINDING DNA BINDING BLAST_(—) PROTEIN FINGER ZINC NUCLEARREPEAT PRODOM TRANSCRIPTION REGULATION PD001562: V22- E84 ZINC FINGERDNA BINDING PROTEIN METAL BLAST_(—) BINDING NUCLEAR ZINC FINGER PRODOMTRANSCRIPTION REGULATION REPEAT PD000072: K449-C512, K365-C428 ZINCFINGER PROTEIN 186 ZINC FINGER BLAST_(—) METAL BINDING DNA BINDINGNUCLEAR PRODOM PD048826: M223-P262 KRAB BOX DOMAIN DM00605 BLAST_DOMO|I48689|11-85: E20-L87 |P51786|24-86: V22-W81 |P51523|5-79: E20-I82|P17097|1-76: L19-E84 ATP/GTP-binding site motif A (P-loop): G340-T347,MOTIFS A536-T543 Zinc finger, C2H2 type, domain: C313-H333, C341- MOTIFSH361, C369-H389, C397-H417, C425-H445, C453- H473, C481-H501, C509-H52914 6259308CD1 264 S15 S25 S118 S125 N23 RNA recognition motif. (a.k.a.RRM, RBD, or: L115- HMMER_PFAM S149 S190 S246 I185 T138 T222 T226PROTEIN NUCLEAR RIBONUCL PD02784: A107- BLIMPS_(—) A143, S149-Q191PRODOM TRANSCRIPTIONAL COACTIVATOR ALY ALY BLAST_(—) PD056100: Q50-K114PRODOM PROTEIN F23B2.6 C01F6.5 M18.7 (C. ELEGANS BLAST_(—) PROTEIN)PD016912: K114-R260 PRODOM 15 7504104CD1 611 S32 S48 S102 S130 N184 N214Helix-loop-helix DNA-binding domain: R509-E562 HMMER_PFAM S143 S209 S324N241 N278 S455 S468 S490 S541 T12 T75 T81 T104 T393 T423 Y77 Myc-type,‘helix-loop-helix’ dimerization domain BLIMPS_(—) proteins BL00038:E517-G532, D542-E562 BLOCKS Myc-type, ‘helix-loop-helix’ dimerizationdomain PROFILESCAN signature: A527-E582 PROTEIN TRANSCRIPTION DNABINDING BLAST_(—) REGULATION NUCLEAR FACTOR PRODOM ALTERNATIVEIMMUNOGLOBULIN SPLICING ITF2 PD005047: Q323-E508; M220-L322 PROTEINTRANSCRIPTION DNA BINDING BLAST_(—) FACTOR REGULATION NUCLEAR PRODOMALTERNATIVE IMMUNOGLOBULIN SPLICING ITF2 PD006397: M25-T222 PROTEINTRANSCRIPTION FACTOR BLAST_(—) REGULATION DNA BINDING NUCLEAR PRODOMALTERNATIVE IMMUNOGLOBULIN SPLICING ITF2 PD006396: F151-P219 PROTEINTRANSCRIPTION DNA BINDING BLAST_(—) REGULATION NUCLEAR FACTOR PRODOMALTERNATIVE IMMUNOGLOBULIN SPLICING ENHANCER PD005628: Q563-M611 HUMANTRANSCRIPTION FACTOR 3 DM01610 BLAST_DOMO |P15881|232-507: L197-K473|Q99081|269-540: L197-D475 |S23391|260-513: L197-D471 MYC-TYPE,‘HELIX-LOOP-HELIX’ BLAST_DOMO DIMERIZATION DOMAIN DM00051|P15881|509-637: L474-H607 Myc-type, ‘helix-loop-helix’ dimerization domain MOTIFSsignature: T546-L561 16 7504121CD1 386 S42 S139 S313 N59 N128 HMG (highmobility group) box: I271-S339 HMMER_PFAM S339 S366 T78 T159 T247 T294T369 Y102 TRANSCRIPTION PROTEIN DN PD02448: N276- BLIMPS_(—) R314,E315-G362 PRODOM FACTOR TRANSCRIPTION PROTEIN BLAST_(—) LYMPHOIDENHANCER BINDING PRODOM DNABINDING NUCLEAR REGULATION 3DSTRUCTUREPD009503: M1-M126 FACTOR TRANSCRIPTION PROTEIN BLAST_(—) DNABINDINGNUCLEAR REGULATION PRODOM LYMPHOID ENHANCER BINDING 3DSTRUCTUREPD007419: N128-P245 FACTOR TRANSCRIPTION LYMPHOID BLAST_(—) ENHANCERBINDING DNABINDING NUCLEAR PRODOM PROTEIN REGULATION 3DSTRUCTUREPD155139: A234-I271 do LEF-1S; DM03940 BLAST_DOMO |P27782|114-284:M116-P220 |S42128|1-171: M116-P220 HMG BOX DM00056 BLAST_DOMO|S42128|173-246: Q260-L334 |P27782|286-359: Q260-L334 17 5635695CD1 807S5 S71 S119 S289 N361 N417 Pyrokinins proteins BL00539: F528-L532BLIMPS_(—) S291 S322 S372 N496 N501 BLOCKS S373 S386 S503 N566 N723 S512S522 S538 N735 S545 S558 S622 S650 T43 T156 T203 T215 T220 T246 T257T467 T544 T567 T674 Y231 18 7503983CD1 257 S69 S82 S138 T49 N67 EF-1guanine nucleotide exchange domain: K171- HMMER_PFAM T123 T240 Y18 I257Elongation factor 1 beta/beta′/delta chain proteins BLIMPS_(—) BL00824:F242-I257, L70-L84, D128-A147, K161- BLOCKS Q198, L199-D233 ELONGATIONFACTOR PROTEIN BLAST_(—) BIOSYNTHESIS 1BETA EF1BETA PRODOMPHOSPHORYLATION 1DELTA EF1DELTA BETA PD002592: E129-I257 ELONGATIONFACTOR 1DELTA EF1DELTA BLAST_(—) PROTEIN BIOSYNTHESIS P36 FACTOR1 DELTAPRODOM FACTOR1D PD010654: A36-P103, L6-G42 ELONGATION FACTOR 1BETA/BETA′/DELTA BLAST_DOMO CHAIN DM01052|P29692|97-280: V73-I257DM01052|P29693|75-264: V73-I257 DM01052|P29522|20-220: T105-I257DM01052|P32192|26-236: Q106-1257 Elongation factor 1 beta/beta′/deltachain signature 1: MOTIFS E129-G137 Elongation factor 1 beta/beta′/deltachain signature 2: MOTIFS V246-I257 Leucine zipper pattern: L56-L77,L63-L84, L70-L91 MOTIFS 19 7503476CD1 113 S95 T8 N58 Ribosomal proteinL15: K75-G107 HMMER_PFAM Ribosomal protein L15 signature: K75-A114PROFILESCAN Ribosomal protein L15 proteins BL00475: K7-R21, BLIMPS_(—)K27-G36, I66-L82, P86-G107 BLOCKS RIBOSOMAL PROTEIN L27A 60S L29 L28 L22BLAST_(—) CRP1 YL24 RP62 PD002840: M1-Y48 PRODOM RIBOSOMAL PROTEIN L15BLAST_DOMO DM00524|S55914|3-145: H19-L111, S3-Y48 DM00524|P41092|4-146:R6-Y48, R12-A55 DM00524|P49637|3-143: S3-Y48, R4-L111DM00524|P48160|3-145: H19-L111, S3-Y48 Ribosomal protein L15 signature:K75-G106 MOTIFS 20 7504023CD1 204 S194 T8 T40 T93 60s Acidic ribosomalprotein: L104-D204 HMMER_PFAM RIBOSOMAL PROTEIN ACIDIC P0 60S BLAST_(—)PHOSPHORYLATION L10E HOMOLOG L10 PRODOM ISOLOG PD002352: V56-V124 RATACIDIC RIBOSOMAL PROTEIN P0 BLAST_DOMO DM00904|P19889|1-315: N34-L104,G105-F203, M1- M50 DM00904|P50346|1-318: G105-F203, I29-L104, P2- G78DM00904|P05317|1-310: L21-G117, I103-F203, K10- V56DM00904|P22685|1-303: G105-F203, K10-G117, K10-G36 21 7504128CD1 144 T18T38 T84 T107 N82 Ribosomal protein L11: L35-D123, V13-P34 HMMER_PFAMRibosomal protein L11 proteins BL00359: N90- BLIMPS_(—) D123, L35-K75BLOCKS Ribosomal protein L11 signature: V89-A143 PROFILESCAN RIBOSOMALPROTEIN L11 BLAST_DOMO DM00681|P30050|6-149: L35-D129, D6-K40DM00681|P17079|6-149: E21-D129 DM00681|P54030|1-143: L35-D129, K11-Q44Ribosomal protein L11 signature: K109-D123 MOTIFS 22 4529338CD1 355 S6S11 S150 S210 N80 N84 QUAKING PROTEIN HOMOLOG KH DOMAIN BLAST_(—) S313T87 T131 N223 RNA BINDING QKI7B QKI7 PD032709: D228- PRODOM T178 T225T300 L325 PROTEIN PHOSPHOPROTEIN P62 ZFM1 BLAST_(—) TYROSINE PUTATIVETRANSCRIPTION PRODOM FACTOR NUCLEAR GAPASSOCIATED PD149659: P100-Q159PROTEIN ZFM1 PUTATIVE PHOSPHOPROTEIN BLAST_(—) P62 TRANSCRIPTION FACTORNUCLEAR KH PRODOM RNA PD002056: R161-R227 PROTEIN KH RNA BINDING QUAKINGBLAST_(—) FEMALE GERMLINE-SPECIFIC TUMOR PRODOM SUPPRESSOR GLD1PD008249: E28-D96 PHOSPHOPROTEIN; P62; GAP; RAS-GAP BLAST_DOMODM02127|A38219|82-278: K32-G224 DM02127|I49140|82-278: K32-G224DM02127|P42083|473-667: N119-R227 DM02127|S64953|79-278: P95-P234 237503460CD1 143 S28 S29 T23 Surp module: R50-E103 HMMER_PFAM SPLICINGPROTEIN MRNA NUCLEAR FACTOR BLAST_(—) SPLICEOSOME REPEAT PREMRNA PRP21PRODOM PUTATIVE PD009917: E48-P129 SPLICEOSOME ASSOCIATED PROTEIN 114SAP BLAST_(—) SF3A120 MRNA PROCESSING SPLICING PRODOM NUCLEAR REPEATPD125875: T17-P47 24 5466630CD1 1048 S60 S62 S68 S170 N214 N229DEAD/DEAH box helicase: R208-E324, E149-T173 HMMER_PFAM S186 S206 S216S401 S456 S545 S713 S722 S740 S888 T7 T54 T284 T301 T338 T592 T699 T879Y873 Helicase conserved C-terminal domain: E471-E567 HMMER_PFAM DEAH-boxsubfamily ATP-dependent helicases BLIMPS_(—) proteins BL00690:G166-Q175, T195-E212, V260- BLOCKS S269 DEAD and DEAH box familiesATP-dependent PROFILESCAN helicases signatures: I236-A287 COSMID 30B8PUTATIVE ATP-DEPENDENT BLAST_(—) RNA HELICASE C06E1.10 CHROMOSOME IIIPRODOM PROTEIN PD041384: K731-H1048 POLYPROTEIN PROTEIN HELICASE GENOMEBLAST_(—) RNA CONTAINS: NUCLEAR ENVELOPE ATP- PRODOM BINDINGNONSTRUCTURAL PD000440: V481- A578, G84-P311, P501-L574 PUTATIVEATP-DEPENDENT RNA HELICASE BLAST_(—) PROTEIN ATP-BINDING RNA-BINDINGPRODOM C06E1.10 CHROMOSOME III PD001244: S325- K416 HELICASE RNAATP-BINDING PROTEIN ATP- BLAST_(—) DEPENDENT NUCLEAR SPLICING MRNAPRODOM PROCESSING PREMRNA PD001259: C571-E716 DEAH-BOX SUBFAMILYATP-DEPENDENT BLAST_DOMO HELICASES DM00649|P34305|227-973: E135-K881DM00649|S53058|382-1163: E135-P854, D802-I876 DM00649|A56236|555-1160:E138-L380, P473- E716, R843-Y883, P807-D826, Q553-M603DM00649|P34498|432-1038: M136-K382, G470- F704, Q764-Y883ATP/GTP-binding site motif A (P-loop): G166-T173 MOTIFS 25 7503474CD1294 S130 S269 T101 N247 N263 Signal_cleavage: M1-G60 SPSCAN T123 T168T183 T233 Sir2 family: D66-P160, G52-R65 HMMER_PFAM PROTEIN SIR2TRANSCRIPTION REGULATION BLAST_(—) REPRESSOR DNA-BINDING ZINC-FINGERPRODOM NUCLEAR REGULATORY SILENT PD002659: P26-L208 Aminotransferasesclass-II pyridoxal-phosphate MOTIFS attachment site: T106-A115 267503498CD1 280 S54 S76 S85 S142 RIBONUCLEASE P PROTEIN SUBUNIT P40BLAST_(—) S151 S166 T40 PD182342: I27-P280, M1-G30 PRODOM T103 T130 Y25627 7504119CD1 288 S4 S124 S159 N53 N157 Ribosomal RNA adeninedimethylase: Q35-V228 HMMER_PFAM S273 T9 T17 T44 T114 Y286 TRANSFERASEMETHYLTRANSFERASE RRNA BLAST_(—) RESISTANCE PROTEIN ADENINE ANTIBIOTICPRODOM N6METHYLTRANSFERASE B PLASMID PD000922: Q35-E276 RIBOSOMAL RNAADENINE DIMETHYLASES BLAST_DOMO DM00429|P37468|16-288: K31-E280DM00429|P44749|5-270: A29-L264 DM00429|P06992|5-265: K104-L264DM00429|P47701|1-255: I138-R265, A30-N157 Immunoglobulins and majorhistocompatibility MOTIFS complex proteins signature: Y202-H208 2871532805CD1 244 S39 S42 S145 T26 Ribosomal protein L30p/L7e: HMMER_PFAMT101 Y151 K84-V136 Ribosomal protein L30 BL00634: V89-G139 BLIMPS_(—)BLOCKS Ribosomal protein L30 signature: PROFILESCAN V88-A137 RIBOSOMALPROTEIN 60S L7 MULTIGENE BLAST_(—) FAMILY RNABINDING REPEAT L7A L7BPRODOM PD149881 A137-R242, PD005715: K7-E82 RAT RIBOSOMAL PROTEIN L7DM02153 BLAST_DOMO P11874|30-245: F47-N244, P25457|33-249: A32- M243,P05737|25-242: E30-N244, P11874|30-245: F47-N244 Ribosomal L30 Motif:I104-V135 MOTIFS Eukaryotic thiol (cysteine) proteases histidine activeMOTIFS site: L186-H196 29 5502992CD1 1953 S9 S21 S58 S131 N46 N440Helicase conserved C-terminal domain: HMMER_PFAM S335 S373 S568 N589N663 D535-G619 S572 S644 S658 N734 N893 S671 S739 S792 N1049 S796 S804S881 S914 S937 S978 S999 S1093 S1131 S1315 S1380 S1418 S1441 S1443 S1465S1466 S1470 S1545 S1550 S1551 S1595 S1786 S1787 S1929 S1934 S1947 T35T244 T252 T297 T355 T419 T459 T591 T711 T770 T777 T782 T847 T995 T1019T1118 T1171 T1362 T1395 T1409 T1576 T1587 T1900 Y118 Y1068 SNF2 andothers N-terminal domain Y186-F473 HMMER_PFAM Chromo domain proteinsBL00598: Y118-V139 BLIMPS_(—) BLOCKS ATP-Binding Nucleoside PD02191:C316-C330, BLIMPS_(—) L336-H364, C596-S621 PRODOM O61845_CAEEL //T04D1.4 PROTEIN PD145655: BLAST_(—) L679-K1031, W1170-S1328,W1111-T1171, P1527- PRODOM W1548 (142) NTP1(5) O22731(3) CHD1(3) //PROTEIN BLAST_(—) HELICASE ATPBINDING NUCLEAR PRODOM DNABINDINGZINCHNGER DNA TRANSCRIPTION REPAIR I PD000441: K385- I473, S304-Q389,Y186-E224, Y177-E249, L235- S267, I667-K696 O61845_CAEEL // T04D1.4PROTEIN PD126894: BLAST_(—) E36-S174, V5-E39 PRODOM HELICASE ATP-BINDINGRNA-BINDING BLAST_(—) INITIATION FACTOR ATP-DEPENDENT PRODOM EUKARYOTICBIOSYNTHESIS DNA-BINDING PD000085: N203-L366 N203-L366 ATP NP_BINDDM00266|P51531|741-1166: I205- BLAST_DOMO V626 DM00266|P32657|397-815ATP NP_BIND I205- V626 DM00266|P40201|500-906 ATP NP_BIND C204- V626DM00266|P28370|126-540 ATP NP_BIND I205- V626 Cell attachment sequence:MOTIFS R867-D869, R1144-D1146 30 7503828 1099 S115 S219 S224 N46 N173Myb-like DNA-binding domain myb_DNA_binding: HMMER_PFAM S267 S273 S286N398 N416 T598-L642 S302 S327 S367 N454 N857 S682 S695 S754 S806 S810S813 S841 T22 T45 T166 T247 T276 T363 T376 T378 T391 T472 T583 T602 T631T726 T743 T744 T847 T911 Y648 Y922 PD025015 O76489(1) P97496(1)Q92922(1) // A BLAST_(—) SWI/SNF ASSOCIATED COMPLEX SUBUNIT PRODOMBRAHMA PROTEIN RELATED MATRIX ACTIN R4-E337 PD007613 // PROTEIN SWI/SNFCOMPLEX BLAST_(—) SUBUNIT A BAF170 CHROMOSOME I PRODOM ASSOCIATEDSIMILAR D338-A554 PD023971 O76489(1) P97496(1) Q92922(1) // A BLAST_(—)SWI/SNF ASSOCIATED COMPLEX SUBUNIT PRODOM BRAHMA PROTEIN RELATED MATRIXACTIN V688-L858 PD006967 // PROTEIN SWI/SNF COMPLEX BLAST_(—) SUBUNIT ABAF170 CHROMOSOME I PRODOM ASSOCIATED SIMILAR Q551-H638 FIBRILLARCOLLAGEN CARBOXYL- BLAST_(—) TERMINAL DM00019|P17656|108-273 Q960-PRODOM G1090 DM00019|P34687|106-271 Q959-P1098 DM00019|P08124|103-269Q959-S1088, P963- P1098, P963-P1097 PROLINE-RICH PROTEINDM03894|A39066|1-159 BLAST_(—) L939-Q1099 PRODOM 31 2647325CD1 203 S78S106 S180 Zinc finger, C2H2 type: HMMER_PFAM F68-H90, Y37-H59,Y166-H188, F96-H118, H124- H146 signal_cleavage: SPSCAN M1-G16 Zincfinger, C2H2 type BL00028: C168-H184 BLIMPS_(—) BLOCKS ProteinZinc-finger metal binding domain PD00066: BLIMPS_(—) H114-C126 PRODOMZINC-FINGER DNA-BINDING METAL-BINDING BLAST_(—) NUCLEAR TRANSCRIPTIONREPEAT PRODOM REGULATION FACTOR PD017719 P36-G191, PD000072: F68-C129ZINC FINGER, C2H2 TYPE, DOMAIN BLAST_DOMO DM00002|P08042|314-358:C73-H118, P17097|353- 390: Q87-K122 Zinc finger, C2H2 type, domain:MOTIFS C39-H59, C70-H90, C98-H118, C126-H146, C168- H188 32 7495416CD1317 S101 S129 T77 Zinc finger, C2H2 type: HMMER_PFAM T142 T306 T308Y119-H141, H63-H85, F91-H113, Y203-H225, Y147- Y147 H169, Y175-H197,H231-H253, Y259-H281 Zinc finger, C2H2 type BL00028: C121-H137BLIMPS_(—) BLOCKS C2H2 type Zinc finger signature PR00048: P90-K103,BLIMPS_(—) L190-G199 PRINTS Protein Zinc-finger metal binding domainPD00066: BLIMPS_(—) H165-C177 PRODOM ZINC-FINGER DNA-BINDINGMETAL-BINDING BLAST_(—) NUCLEAR PATERNALLY EXPRESSED PRODOM PD017719:A60-F296, G87-H281, G115-F296 ZINC-FINGER DNA-BINDING METAL-BINDINGBLAST_(—) NUCLEAR TRANSCRIPTION REPEAT PRODOM REGULATION FACTORPD000072: R117-C180 R89-C152, R61-C124, K173-C236, R201-C264, K145-C208ZINC FINGER, C2H2 TYPE, DOMAIN BLAST_DOMO DM00002|P17097|353-390:R194-K229 DM00002|Q05481|789-829: R167-D207, R194-C233, R83-E123DM00002|P08042|314-358 C68-H113, C96-H141 DM00002|Q05481|831-885C180-R232 Zinc finger, C2H2 type, domain: MOTIFS C65-H85, C93-H113,C121-H141, C149-H169, C177- H197, C205-H225, C233-H253, C261-H281 338096177CD1 579 S9 S91 S131 S156 N2 N40 Zinc finger, C2H2 type:HMMER_PFAM S161 S189 S254 N108 N149 Y520-H542, Y548-H570, Y408-H430,Y464-H486, S260 S330 S362 N159 N258 F380-H402 S558 T18 T52 N357 N360T231 T250 T320 N394 N450 T388 T528 Y270 KRAB box: V8-E70 HMMER_PFAM Zincfinger, C2H2 type BL00028: C410-H426 BLIMPS_(—) BLOCKS C2H2 type Zincfinger signature PR00048: P407- BLIMPS_(—) S420, L535-G544 PRINTSPROTEIN Zinc Finger PD01066 F10-G48 BLIMPS_(—) PRODOM ProteinZinc-finger metal binding domain PD00066: BLIMPS_(—) H398-C410 PRODOMZINC-FINGER DNA-BINDING METAL-BINDING BLAST_(—) NUCLEAR TRANSCRIPTIONREPEAT PRODOM REGULATION FACTOR PD000072: K434-C497, K490-C553,K406-C469, K518-H570, K378-C441, K462-C525; PD001562: V8-E70; PD033163:D353- K490, C382-K518, V481-V574 ZINC-FINGER DNA-BINDING METAL-BINDINGBLAST_(—) NUCLEAR PATERNALLY EXPRESSED PRODOM PD017719: L340-H570,G376-V573, G348-H570, P269-K518, N295-H542 KRAB BOX DOMAIN BLAST_DOMODM00605|P52736|1-72: V8-C77 DM00605|I48689|11-85: Q5-K71DM00605|P51523|5-79: Q5-F79 DM00605|P51786|24-86: V8-W67 ATP/GTP-bindingsite motif A (P-loop): G271-S279 MOTIFS Zinc finger, C2H2 type, domain:MOTIFS C382-H402, C410-H430, C438-H458, C466-H486, C494-H514, C522-H542,C550-H570 34 666763CD1 730 S68 S100 S102 N244, N283, MCM family proteinsBL00847H: T151-R168, A42- BLIMPS_(—) S185 S206 S263 N310, N365, K96,P123-Q142 BLOCKS S278 S314 S318 N449, N611, S380 S389 S404 N632 S450S477 S521 S539 S543 S631 S634 S686 S690 S730 T28 T79 T124 T152 T176 T313T354 T511 T547 T697 T709 REPLICATION DNA CELL DNA-BINDING BLAST_(—)REGULATION ATP-BINDING TRANSCRIPTION PRODOM NUCLEAR FACTOR LICENSINGPD001041: R27- K104, M108-M191 MCM2/3/5 FAMILY BLAST_DOMODM00603|JC4580|223-719: R27-G198 DM00603|P34647|193-736 L34-M191DM00603|P33991|340-862 R27-D179, T679-K704 DM00603|P30665|386-928R27-V254 35 7504091CD1 315 S66 S94 S127 T47 N30 N147 Ribosomal proteinL3: HMMER_PFAM T142 T174 T203 N253 K124-K267, K103-Q123 signal_cleavage:SPSCAN M1-G43 Ribosomal protein L3 proteins BL00474: L99-L109,BLIMPS_(—) F165-G199, G208-N244 BLOCKS Ribosomal protein L3 signature:PROFILESCAN F146-A209 RIBOSOMAL L3 MITOCHONDRIAL 60S BLAST_(—)MITOCHONDRION PD105243: M1-G29; PRODOM PD036323 N30-Q123, K124-E134;PD036320: I250- A315, PD002374: F131-I249, K246-K267 RIBOSOMAL PROTEINL3 BLAST_DOMO DM00364|P38515|9-200: K112-K267 DM00364|P09001|105-300V119-D268, G105-Q123 DM00364|P49404|87-295 E130-D268, G105-Q123DM00364|P31334|68-263 G114-D268 Ribosomal protein L3 signature: MOTIFSF165-R188 36 7503568 317 S35 S154 S217 N11 N48 KH domain: R101-G150,E243-G291, R17-G63 HMMER_PFAM S222 T15 T99 N89 N140 T142 T240 T283 KHdomain proteins family PF00013: L112-I123 BLIMPS_(—) PFAM PROTEINNUCLEAR RNABINDING BLAST_(—) RIBONUCLEOPROTEIN DNABINDING REPEAT PRODOMHNRNPE1 POLYCBINDING NUCLEIC ACID PD010726: L194-T240, I151-Q193 RNABINDING PROTEIN PUTATIVE PRE MRNA BLAST_(—) SPLICING FACTOR PD182839:L14-P180 PRODOM PROTEIN NUCLEAR RNABINDING BLAST_(—) RIBONUCLEOPROTEINDNABINDING REPEAT PRODOM HNRNPE1 POLYCBINDING PHOSPHORYLATION PROTEIN1PD151096: P64- R101 KH DOMAIN BLAST_DOMO DM00168|I48281|86-167: S86-E168DM00168|S58529|86-167: S86-E168 DM00168|I48281|6-84: I6-S85 COMPLEX;NUCLEAR; RIBONUCLEOPROTEIN; BLAST_DOMO HETEROGENEOUS;DM08370|S58529|232-328: L194-I289 37 7504101CD1 748 S83 S129 S203 COILCOILED MYOSIN CHAIN ATP-BINDING BLAST_(—) S206 S224 S274 HEAVY FILAMENTMUSCLE REPEAT PRODOM S294 S493 S526 INTERMEDIATE PD000002: S600 S614S616 Q338-I445, E331-K442, Q338-E444 S642 T17 T175 T183 T322 T330 T485CHROMATIN ASSEMBLY FACTORI P150 BLAST_(—) SUBUNIT PD132442: M19-L360;PD096339: I438- PRODOM D601; PD124531: F634-Q721 TROPOMYOSINDM00077|P53935|580-755: R320- BLAST_(—) K442 PRODOMDM00077|Q07283|445-599: K327-E444 DM00077|P37709|1104-1277: T330-R447TRICHOHYALIN DM03839|P37709|632-1103: BLAST_DOMO E331-R447 Cellattachment sequence: MOTIFS R196-D198 38 6946680CD1 609 S24 S34 S64 S80N228 N590 Zinc finger, C2H2 type: HMMER_PFAM S89 S97 S125 S168Y554-H576, Y582-H604, Y386-H408, Y330-H352, S424 S568 T15 F442-H464,Y414-H436, F358-H380, Y302-H324, T158 F498-H520, Y526-H548, Y470-H492KRAB box V14-E76 HMMER_PFAM Zinc finger, C2H2 type, domain proteinsBL00028: BLIMPS_(—) C388-H404 BLOCKS C2H2-type zinc finger signaturePR00048: P413- BLIMPS_(—) R426, L429-G438 PRINTS Protein zinc fingerPD01066 F16-G54 BLIMPS_(—) PRODOM Protein zinc finger PD00066 H376-C388BLIMPS_(—) PRODOM PROTEIN ZINCFINGER METALBINDING BLAST_(—) DNABINDINGZINC FINGER PATERNALLY PRODOM EXPRESSED ZNFINGER PW1 PD017719: G298-H548, F269-H520, G354-F591, G242-H492, G410- E607 ZINCFINGERMETALBINDING DNABINDING BLAST_(—) PROTEIN FINGER ZINC NUCLEAR REPEATPRODOM TRANSCRIPTION REGULATION PD001562: V14- E76 ZINCFINGER DNABINDINGPROTEIN BLAST_(—) METALBINDING NUCLEAR ZINC FINGER PRODOM TRANSCRIPTIONREGULATION REPEAT PD000072: K412-C475, K524-C587, K384-C447, K440-C503,K328-C391, R496-C559, K468-C531 MYELOBLAST METAL-BINDING ZINC-FINGERBLAST_(—) NUCLEAR KIAA0211 DNA-BINDING PD149061: PRODOM E387-N589 KRABBOX DOMAIN DM00605|I48208|18-93: V14- BLAST_DOMO R77DM00605|P52738|3-77: Q11-Q81 DM00605|Q05481|10-83: G12-M79DM00605|P52736|1-72: V14-C84 Zinc finger, C2H2 type, domain: MOTIFSC304-H324, C332-H352, C360-H380, C388-H408, C416-H436, C444-H464,C472-H492, C500-H520, C528-H548, C556-H576, C584-H604 39 7001142CD1 536S24 S34 S85 S108 N341 N453 Zinc finger, C2H2 type: HMMER_PFAM S124 S198S256 Y498-H520, Y330-H352, Y386-H408, Y246-H268, S340 S368 T15 T97Y358-H380, Y470-H492, Y414-H436, L218-H240 T152 T179 T194 Y274-H296,Y302-H324 Y442-H464 T506 KRAB box: V14-K76 HMMER_PFAM Zinc finger, C2H2type, domain proteins BL00028: BLIMPS_(—) C388-H404 BLOCKS PROTEIN ZINCFINGER ZINC PD01066: F16-G54 BLIMPS_(—) PRODOM PROTEIN BOLA TRANSCRIPTIPD02462: T325- BLIMPS_(—) E359, V290-E303 PRODOM PROTEIN ZINCFINGERMETALBINDING BLAST_(—) DNABINDING ZINC FINGER PATERNALLY PRODOMEXPRESSED ZNFINGER PW1 PD017719: G270- H520, G242-H492, C220-H464,G326-E523, K181- H408; PD001562: V14-K76; D000072: K440-C503, K328-C391,K244-C307, K412-C475, R384-C447, K356-C419, K300-C363, K272-C335,K216-C279 ZINC FINGER PROTEIN ZINCFINGER BLAST_(—) METALBINDINGDNABINDING PUTATIVE PRODOM REX2 TRANSCRIPTION REGULATION PD033163:E224-K356 KRAB BOX DOMAIN DM00605|I48208|18-93: V14- BLAST_DOMO R77DM00605|P52738|3-77: Q11-S85 DM00605|Q05481|10-83: L13-R77DM00605|Q03923|1-75: V14-R77 Zinc finger, C2H2 type, domain: MOTIFSC220-H240, C248-H268, C276-H296, C304-H324, C332-H352, C360-H380,C388-H408, C416-H436, C444-H464, C472-H492, C500-H520 40 71158380CD1 643S295 S351 S379 N12 KRAB box: V4-D54 HMMER_PFAM S435 T14 T36 T142 T164T276 T282 T302 T508 Zinc finger, C2H2 type: Y560-H582, Y225-H247,HMMER_PFAM Y309-H331, H337-H359, Y253-H275, Y476-H498, Y169-Q191,Y449-H470, Y131-H163, Y588-H610, Y621-H643, H393-H415, Y281-H303,Y197-H219, Y421-H443, Y504-H526, Y365-H387, Y532-H554 Zinc finger, C2H2type, domain proteins BL00028: BLIMPS_(—) C171-H187 BLOCKS C2H2-typezinc finger signature PR00048: P475- BLIMPS_(—) F488, L491-G500 PRINTSPROTEIN ZINC-FINGER META PD00066: H494- BLIMPS_(—) C506 PRODOM PROTEINZINC FINGER ZINC PD01066: L6-G44 BLIMPS_(—) PRODOM PROTEIN ZINCFINGERMETALBINDING BLAST_(—) DNABINDING ZINC FINGER PATERNALLY PRODOMEXPRESSED ZNFINGER PW1 PD017719: P308- F541, G361-F597, G221-D459,P420-H643, G389- F630, G137-H387, G165-H415 ZINCFINGER DNABINDINGPROTEIN BLAST_(—) METALBINDING NUCLEAR ZINC FINGER PRODOM TRANSCRIPTIONREGULATION REPEAT PD000072: K279-C342, K419-C481, P587-H643, K474-C537,R556-C626, K307-C370, K167-C230, K251-C314, P336-C398, K530-C593,P196-C258, K363-C426 KRAB BOX DOMAIN DM00605|P52737|1-76: M1- BLAST_DOMOD76 DM00605|I49636|10-85: S3-D54 ZINC FINGER, C2H2 TYPE, DOMAINBLAST_DOMO DM00002|Q05481|789-829: E412-K452, E272-Q313, R495-E536 Cellattachment sequence: R192-D194 MOTIFS Zinc finger, C2H2 type, domain:C199-H219, C227- MOTIFS H247, C255-H275, C283-H303, C311-H331, C339-H359, C367-H387, C395-H415, C423-H443, C478- H498, C506-H526, C534-H554,C562-H582, C590- H610, C623-H643 41 7503861CD1 1143 S59 S74 S119 S145N72 N759 Zinc finger, C2H2 type: HMMER_PFAM S214 S251 S318 F1000-H1022,Y738-H755, V615-H638, Y889-H912, S322 S328 S331 F768-H791, A1029-H1052,H703-H726, Y919-H945, S334 S342 S348 W859-H882, Y675-H698, Y644-C666,Y587-H612 S357 S383 S717 S805 S811 S836 S903 S935 S992 S1011 S1081 T22T32 T73 T300 T339 T379 T479 T520 T860 T885 T914 T955 T993 Zinc finger,C2H2 type, domain proteins BL00028: BLIMPS_(—) C891-H907 BLOCKS PROTEINZINC-FINGER TALBINDING BLIMPS_(—) DNABINDING PD00066: H634-C646 PRODOMMYELOBLAST KIAA0211 ZINCFINGER BLAST_(—) METALBINDING DNABINDING PRODOMPD178887: N949-V1143; PD185235: M1-G586; PD149061: C589-L886 Zincfinger, C2H2 type, domain: MOTIFS C705-H726, C770-H791, C861-H882,C891-H912, C1031-H1052 42 7758395CD1 1099 S38 S94 S346 S350 N209 N883Myb-like DNA-binding domain: S830-K875 HMMER_PFAM S358 S438 S453 N977S461 S486 S494 S572 S596 S645 S761 S798 S898 S910 S923 S962 S979 S1002S1012 S1019 S1059 T454 T499 T513 T568 T573 T687 T786 T828 T873 T887 T893T1009 T1025 Y46 Y654 Zinc finger, C2H2 type, domain: MOTIFS C1040-H106043 71039312CD1 1006 S52 S67 S79 S130 N329 N735 Zinc finger, C2H2 type:F668-H692, Y551-H575, HMMER_PFAM S156 S163 S173 F610-H634, Y490-H515S205 S213 S231 S237 S258 S310 S337 S374 S433 S504 S530 S564 S687 S720S721 S732 S861 S899 T181 T196 T225 T259 T332 T367 T445 T452 T503 T579T620 T715 Y346 Y656 FINGER ZINC DM04988 BLAST_DOMO |JH0797|457-514:H571-I627 |JH0797|396-455: I509-V568 |JH0797|516-572: E628-M685ATP/GTP-binding site motif A (P-loop): G541-S548 MOTIFS Zinc finger,C2H2 type, domain: C553-H575, C612- MOTIFS H634, C670-H692 44 7291318CD1768 S15 S27 S239 S313 N19 signal_cleavage: M1-A34 SPSCAN S410 S416 S428S446 S464 S539 S607 T75 T154 T219 T222 T285 T338 T403 T657 Y417 Zincfinger, C2H2 type: Y359-H381, F480-H502, F58- HMMER_PFAM C81, F625-G648,Y564-Q591, L236-H258, Y536- H558, H597-H619, F264-H286, Y417-H439, F508-H530, Y387-H411, Y454-H478 Zinc finger, C2H2 type, domain proteinsBL00028: BLIMPS_(—) C482-H498 BLOCKS Cytidine and deoxycytidylatedeaminases zinc-binding BLIMPS_(—) regions BL00903: A476-C485 BLOCKSPROTEIN ZINC-FINGER META PD00066: H254- BLIMPS_(—) C266 PRODOM Zincfinger, C2H2 type, domain: C238-H258, C266- MOTIFS H286, C361-H381,C389-H411, C419-H439, C456- H478, C482-H502, C510-H530, C538-H558, C599-H619 45 2638619CD1 561 S3 S27 S32 S71 N69 N177 ELM2 domain: K211-P272HMMER_PFAM S119 S148 S157 S158 S197 S249 S319 S349 S363 S395 S459 S483S501 S521 T167 T199 T386 T476 Y236 Y259 Y288 Y356 Y368 Myb-likeDNA-binding domain: L315-K361 HMMER_PFAM ER1 PD126939: E42-Q285BLAST_(—) PRODOM PROTEIN METASTASIS-ASSOCIATED MTA1 BLAST_(—) SIMILARMTA1 T27C4.4 KIAA0458 C04A2.2 PRODOM CHROMOSOME II PD011563: A286-R37746 2810014CD1 123 S23 S53 S63 S70 N80 N113 signal_cleavage: M1-A29SPSCAN S77 S97 T57 T84 47 3457155CD1 1236 S10 S18 S45 S97 N299 N704LYASE PROTEIN PHYCOBILIS PD01642 S874- BLIMPS_(—) S186 S318 S333 E902PRODOM S337 S342 S348 S364 S365 S449 S470 S485 S728 S846 S872 S892 S917S929 S1019 S1096 S1166 S1209 T16 T86 T135 T494 T581 T1031 T1079 T1159T1190 Y566 Y1095 TB-Binding Protein TIP120 PD044220 A4-D1223 BLAST-PRODOM Leucine zipper pattern: L155-L176 MOTIFS 48 7435171CD1 357 S32S135 S147 N294 signal_cleavage: M1-A67 SPSCAN S171 S180 S185 S244 S253S255 S314 T149 T230 Homeobox domain: K228-R284 HMMER_PFAM Homeobox′domain proteins BL00027: L242-R284 BLIMPS_(—) BLOCKS Homeobox′antennapedia-type protein BL00032: BLIMPS_(—) A198-E220, R231-T269,Q270-A287 BLOCKS Homeobox′ domain signature and profile: Q241-V304PROFILESCAN Homeobox signature PR00024: K249-L260, L264- BLIMPS_(—)W274, W274-K283 PRINTS PROTEIN HOMEOBOX DNA-BINDING BLAST_(—) NUCLEARNKX5.1 DEVELOPMENTAL PRODOM HOMEODOMAIN NKX51 PD034587: F83-R226PD019212: L286-V357 PROTEIN HOMEOBOX DN-BINDING NUCLEAR BLAST_(—)DEVELOPMENTAL TRANSCRIPTION PRODOM REGULATION FACTOR HOMEODOMAINMETAL-BINDING PD000010: R226-Q285 HOMEOBOX DM00009 BLAST_DOMO|P42581|325-388: P223-A287 |I48690|325-388: P223-A287 |A47234|192-259:R226-A287 |B41224|153-215: R226-Q285 Homeobox′ domain signature:L260-K283 MOTIFS 49 7499936CD1 168 S20 T151 signal_cleavage: M1-A43SPSCAN Ligand-binding domain of nuclear hormone: G10- HMMER_PFAM L161Retinoic acid receptor signature PR00545: N12-G29, BLIMPS_(—) F52-E72,P92-Y109, K113-R132, E140-E159 PRINTS RECEPTOR PROTEIN NUCLEAR BLAST_(—)TRANSCRIPTION REGULATION DNA-BINDING PRODOM ZINC FINGER HORMONE FAMILYMULTIGENE PD000112: L7-I134 RECEPTOR TRANSCRIPTION REGULATION BLAST_(—)DNA-BINDING NUCLEAR PROTEIN ZINC PRODOM FINGER RETINOIC ACID MULTIGENEPD149760: G135-H165 NUCLEAR HORMONES RECEPTORS DNA- BLAST_DOMO BINDINGREGION DM00047 |Q05343|130-391: L7-A93 |P28700|130-391: L7-A93|P19793|125-386: L7-A93 |C41727|130-391: L7-A93 50 7504125CD1 142 S139T27 N50 signal_cleavage: M1-D17 SPSCAN Ets-domain: A4-K69 HMMER_PFAM ETSdomain signature PR00454: I5-Q18, N29-L47, BLIMPS_(—) R48-Y66 PRINTSEts-domain proteins BL00345: M1-K19, K34-S84 BLIMPS_(—) BLOCKSEts-domain signatures and profile: S3-L35, G31- PROFILESCAN T113 ETSDOMAIN PROTEIN NUCLEAR DNA- BLAST_(—) BINDING ACCESSORY FACTOR PRODOMTRANSCRIPTION SERUM RESPONSE ELK4 PD008319: Y65-S142 PROTEIN DNA-BINDINGNUCLEAR BLAST_(—) TRANSCRIPTION FACTOR REGULATION ETS PRODOMPROTO-ONCOGENE ACTIVATOR ALTERNATIVE PD000803: I5-K69 ETS-DOMAINDM02126|P41970|98-406: Y65-S142 BLAST_DOMO ETS-DOMAIN DM00281 BLAST_DOMO|P41970|1-96: M1-K69 |I48680|1-96: M1-K69 ETS-DOMAINDM02126|I48680|98-409: Y65-K141 BLAST_DOMO 51 7505742CD1 477 S91 S216S248 N220 N316 Ets-domain signature 1: L7-L15 MOTIFS S265 S423 S443 N341Ets-domain signature 2: K51-Y66 T191 T258 T266 T267 T286 Fork headdomain: K169-R264 HMMER_PFAM Fork head domain signature PR00053:K169-I182, BLIMPS_(—) L190-R207, W213-V230 PRINTS Fork head domainproteins BL00657: K169-K210, BLIMPS_(—) Q214-G256 BLOCKS Fork headdomain signatures and profile: L101-G194 PROFILESCAN TRANSCRIPTIONFACTOR DNA-BINDING BLAST_(—) NUCLEAR PROTEIN BF1 BRAIN REGULATION PRODOMDEVELOPMENTAL BF1 PD009393: I254-P412 PROTEIN TRANSCRIPTION FACTORNUCLEAR BLAST_(—) DNA-BINDING REGULATION FORK HEAD PRODOM FORKHEADDOMAIN PD000425: K169-R264 TRANSCRIPTION FACTOR BRAIN BLAST_(—)REGULATION DNA-BINDING NUCLEAR PRODOM PROTEIN DEVELOPMENTAL BF1 BF1PD012927: S413-H477 TRANSCRIPTION FACTOR BF1 BRAIN 1 BF1 BLAST_(—) HFK1REGULATION DNA-BINDING NUCLEAR PRODOM PROTEIN DEVELOPMENTAL PD049691:G86- D131 FORK HEAD DNA-BINDING DOMAIN DM00381 BLAST_DOMO|P55315|58-332: P58-L333 |A47446|44-314: H48-L333, H37-K152|P32031|72-344: P122-P326 |P32030|22-301: E142-A278, H50-P58, H52-E94,P45-H54 Fork head domain signature 1: K169-I182 MOTIFS Fork head domainsignature 2: W213-H219 52 7505757CD1 1274 S10 S18 S83 S135 N337 N742LYASE PROTEIN PHYCOBILIS PD01642: S912- BLIMPS_(—) S224 S356 S371 E940PRODOM S375 S380 S386 S402 S403 S487 S508 S523 S766 S884 S910 S930 S955S967 S1057 S1134 S1204 S1247 T16 T124 T173 T532 T619 T1069 T1117 T1197T1228 Y604 Y1133 PUTATIVE TB-BINDING PROTEIN TIP 120 BLAST_(—) PD044220:R61-D1261 PRODOM Leucine zipper pattern: L193-L214 MOTIFS 53 7504126CD191 S24 S89 T2 T55 signal_cleavage: M1-A45 SPSCAN RIBOSOMAL PROTEIN 40SS5 5S PROBABLE BLAST_(—) PD004090: M1-Q36 PRODOM RIBOSOMAL PROTEIN S7DM00334 BLAST_DOMO |P49041|182-209: Q36-R91 |P26783|96-223: Q36-R91 547504099CD1 311 S169 S196 S202 HPBRII4 MRNA BLAST_(—) S220 S231 S248PD112364: M1-P70 PRODOM S266 T163 Y281 PD066177: V116-R165 Y308PD029583: T166-Q233 PD175646: D234-Y281 HPBRII; DM05499|S57447|BLAST_DOMO 356-450: H115-A210 251-354: P56-P114, P57-P148, G67-G158PROLINE-RICH PROTEIN DM03894|P05142|1- BLAST_DOMO 134: P57-P147,V36-G158, P56-P114 FIBRILLAR COLLAGEN CARBOXYL- BLAST_DOMO TERMINALDM00042|A41132|43-133: P56-P124, P58-P142, P56-V116 Cell attachmentsequence: R151-D153 MOTIFS 55 7505733CD1 110 S90 S100 signal_cleavage:M1-L63 SPSCAN Ribosomal protein S24e signature: S21-I75 PROFILESCANPROTEASE ORF DERIVED FROM INTEGRASE BLAST_(—) CODING REGION REGIONS D1LEADER PRODOM PD152194: D20-Q110 56 7959829CD1 176 T6 T11 T51 T88 N4 N9SYNTHASE I PSEUDOURIDYLATE PD02906: BLIMPS_(—) T117 N86 C114-Q126,L130-L164, Y71-E83 PRODOM SYNTHASE; PSEUDOURTOYLATE; TRNA; BLAST_DOMOPSEUDOURIDINE; DM02282 |Q09524|29-297: R62-N165 |P31115|91-343: R63-R16657 7502168CD1 532 S6 S19 S37 S90 N519 chromo′ (CHRromatin OrganizationModifier): E9- HMMER_PFAM S118 S119 S120 I49 S126 S360 S409 S411 S426S438 S459 S465 S467 S469 S498 T84 T176 T188 T281 T294 T408 T454 T489Chromo domain proteins BL00598: E28-I49 BLIMPS_(—) BLOCKS Chromo domainsignature and profile: I17-Q68 PROFILESCAN Chromodomain signaturePR00504: E9-I17, L22- BLIMPS_(—) W36, S37-I49 PRINTS MODIFIER 3 PROTEINM33 NUCLEAR BLAST_(—) TRANSCRIPTION REGULATION REPRESSOR PRODOMPD138310: K131-T506 CHROMO DOMAIN DM00963 BLAST_DOMO |P30658|1-190:M1-R190 |P34618|1-189: G8-I160 |P05205|13-184: E9-R132 |P45973|9-158:S5-K96 Chromo domain signature: Y29-I49 MOTIFS 58 7503888CD1 1492 S2 S78S111 S446 N248 N563 SNF2 and others N-terminal domain: Y757-F1052HMMER_PFAM S655 S660 S662 N1302 S699 S1022 S1058 S1227 S1335 S1366 S1415S1420 S1431 S1472 S1476 S1487 S1489 S442 S624 S833 S850 S1079 S1155S1254 S1255 S1272 S1282 S1322 S1404 S1437 S1262 S1462 S937 T428 T511T629 T858 T1110 T1130 T1141 T1203 T1241 T11 T308 T453 T494 T739 T1129T1229 T1304 Y90 Y718 Y1224 Bromodomain: M1307-V1397, K1140-S1155HMMER_PFAM Helicase conserved C-terminal domain: T1110-G1194 HMMER_PFAMBromodomain proteins BL00633: L918-P930, P1340- BLIMPS_(—) Y1364,D1373-N1385 BLOCKS Bromodomain signature PR00503: Q1325-E1338,BLIMPS_(—) L1339-I1355, I1355-D1373 PRINTS Bromodomain signature andprofile: P1334-S1404 PROFILESCAN I ATP-BINDING NUCLEOSIDE PD02191: Y877-BLIMPS_(—) C891, N898-N926, K997-Y1008, V1171-Q1196 PRODOM PROTEINBROMODOMAIN HELICASE BLAST_(—) NUCLEAR ATP-BINDING TRANSCRIPTION PRODOMREGULATION ACTIVATOR BRAHMA POSSIBLE PD007692: E365-K572 PROTEINHELICASE ATP-BINDING NUCLEAR BLAST_(—) DNA-BINDING ZINC FINGER DNAPRODOM TRANSCRIPTION REPAIR I PD000441: L870- L1035, I932-M1050,N771-E821, Y757-I793, G390- E449, L456-I479, Q460-A509 PROTEIN POSSIBLEGLOBAL TRANSCRIPTION BLAST_(—) ACTIVATOR REGULATION NUCLEAR PRODOMBROMODOMAIN ATP-BINDING HELICASE PD017589: G594-K687 PROTEIN POSSIBLEGLOBAL TRANSCRIPTION BLAST_(—) ACTIVATOR REGULATION NUCLEAR PRODOMBROMODOMAIN ATP-BINDING HELICASE PD151443: E286-V364 BROMODOMAIN DM02887BLAST_DOMO |P51532|177-770: L177-N771 |S45252|177-770: L177-N771|S39059|176-768: L177-N770 ATP NP_BIND DM00266|S45252|772-1200: N772-BLAST_DOMO V1199 Bromodomain signature: S1327-F1384 MOTIFS Leucinezipper pattern: L907-L928 MOTIFS

TABLE 4 Polynucleotide SEQ ID NO:/ Incyte ID/Sequence Length SequenceFragments 59/7503848CB1/ 1-236, 1-522, 4-541, 21-268, 21-662, 29-630,87-618, 131-5006, 147-537, 469-723, 555-861, 555-1022, 668-769, 5007668-770, 668-859, 668-1053, 688-750, 688-765, 688-766, 688-832,688-1041, 693-832, 726-988, 727-840, 728-840, 742-840, 749-846, 757-846,786-832, 791-850, 796-850, 800-840, 818-846, 868-1008, 871-1410,874-958, 874-980, 874-982, 874-996, 874-1062, 875-982, 881-932,881-1069, 881-1248, 886-1053, 902-978, 902-996, 902-1053, 909-1062,910-1056, 942-1041, 942-1181, 955-1041, 995-1032, 995-1062, 1004-1296,1013-1098, 1013-1107, 1013-1112, 1013-1133, 1013-1160, 1013-1177,1013-1189, 1013-1190, 1013-1266, 1014-1053, 1014-1062, 1052-1151,1052-1190, 1052-1266, 1059-1153, 1070-1290, 1080-1321, 1096-1127,1096-1240, 1096-1266, 1096-1446, 1097-1248, 1101-1266, 1137-1240,1172-1240, 1182-1721, 1192-1241, 1253-1283, 1257-1776, 1275-1325,1346-1458, 1354-2012, 1382-1450, 1386-1974, 1449-1708, 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269-340112/7504099CB1/ 1-259, 1-1199, 34-249, 59-259, 65-217, 70-259, 72-179,78-185, 79-239, 87-259, 89-259, 89-525, 89-548, 89-557, 1408 257-488,271-704, 305-578, 305-840, 334-804, 336-626, 341-880, 353-584, 353-585,370-733, 371-660, 411-1082, 424-685, 424-709, 426-684, 434-1000,445-721, 446-700, 450-700, 457-737, 467-699, 477-1022, 483-757, 500-771,505-1183, 508-1133, 512-757, 512-1057, 514-889, 526-754, 526-757,526-760, 526-766, 526-769, 526-770, 526-772, 526-794, 530-716, 541-773,541-838, 541-1215, 558-858, 563-768, 564-786, 582-723, 587-825, 587-837,599-840, 612-852, 614-866, 626-780, 632-915, 644-904, 644-964, 669-907,671-1135, 685-944, 706-897, 706-970, 706-1184, 706-1190, 712-998,713-1011, 714-1006, 721-987, 748-1193, 754-1037, 755-1032, 760-1056,764-1199, 765-950, 765-1021, 780-1193, 790-1162, 794-1079, 826-1165,826-1267, 837-1077, 837-1081, 845-1408, 857-1052, 862-1108, 865-1128,865-1148, 897-1136, 897-1144, 897-1170, 937-1179, 948-1188, 962-1258113/7505733CB1/ 1-600, 321-590, 331-455, 338-600, 347-587, 349-594,358-577, 365-600, 375-766, 386-595, 395-600, 402-602, 444-600, 1363478-590, 478-600, 478-1363, 479-594, 479-602, 656-781, 656-1156,1011-1154, 1144-1346 114/7959829CB1/ 1-103, 1-593, 12-173, 238-738,256-515, 293-484, 327-605, 439-1071 1071 115/7502168CB1/ 1-256, 51-362,81-256, 180-256, 190-235, 190-443, 273-939, 280-1049, 763-1164,763-1420, 931-1561, 1088-1585, 2140 1316-1676, 1617-1981, 1617-2017,1617-2018, 1617-2043, 1617-2048, 1617-2066, 1617-2075, 1617-2104,1619-2013, 1620-2076, 1621-1809, 1623-1977, 1624-2140, 1634-2140116/7503888CB1/ 1-245, 1-685, 5-4956, 20-686, 27-267, 36-277, 44-306,53-356, 55-486, 67-334, 68-729, 74-350, 75-333, 80-380, 4980 110-182,159-933, 165-816, 171-853, 173-460, 173-742, 173-803, 173-1010, 174-441,174-850, 280-880, 411-934, 411-943, 456-671, 460-623, 501-792, 514-867,524-730, 582-876, 590-1045, 602-854, 602-880, 606-1101, 624-843,653-756, 684-1177, 756-999, 927-1153, 983-1481, 991-1405, 1014-1286,1143-1631, 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TABLE 5 Polynucleotide SEQ Representative ID NO: Incyte Project ID:Library 59 7503848CB1 293TF5T01 60 2608080CB1 BRAIFEE05 61 7503402CB1GBLATUT01 62 7503517CB1 PANCNOT05 63 7500014CB1 NERDTDN03 64 7501365CB1HEAONOE01 65 7503540CB1 SCORNON02 66 7504326CB1 BRAUNOR01 67 7504388CB1BRAITUT12 68 2828380CB1 PANCNOE02 69 6456919CB1 LUNLTUT11 70 7502244CB1CONTTUT01 71 7498718CB1 CERVNOT01 72 6259308CB1 KIDEUNE02 73 7504104CB1UTRSDIC01 74 7504121CB1 KIDEUNE02 75 5635695CB1 UTRSTMR01 76 7503983CB1FIBRUNT02 77 7503476CB1 PANCTUT02 78 7504023CB1 COLNPOT01 79 7504128CB1PANCNOT04 80 4529338CB1 HEARNON03 81 7503460CB1 EPIPNOT01 82 5466630CB1COLENOR03 83 7503474CB1 PANCNOT05 84 7503498CB1 ENDCNOT03 85 7504119CB1MUSCNOT10 86 71532805CB1 BRAIFEN03 87 5502992CB1 THYMNOE02 88 7503828CB1BRACNOK02 89 2647325CB1 PROSTME06 90 7495416CB1 UTRCDIE01 91 8096177CB1TESTNON04 92 666763CB1 OVARDIJ01 93 7504091CB1 HNT2RAT01 94 7503568CB1UTRSNOT02 95 7504101CB1 THYRDIE01 96 6946680CB1 BRAENOT02 97 7001142CB1MMLR3DT01 98 71158380CB1 MCLDTXN05 99 7503861CB1 FIBRTXS07 1007758395CB1 LUNGDIS03 101 71039312CB1 BRANDIN01 102 7291318CB1 BRAIFER06103 2638619CB1 COLNFET02 104 2810014CB1 LUNGTUT17 105 3457155CB1THP1NOT03 106 7435171CB1 PANCDIR02 107 7499936CB1 PENITUT01 1087504125CB1 CONNNOT01 109 7505742CB1 KIDEUNE02 110 7505757CB1 THP1NOT03111 7504126CB1 SCORNOT04 112 7504099CB1 KERANOT01 113 7505733CB1TESTTUT02 114 7959829CB1 PROSBPT07 115 7502168CB1 BRAIUNT01 1167503888CB1 NOSEDIC02

TABLE 6 Library Vector Library Description 293TF5T01 pINCY Library wasconstructed using RNA isolated from a transformed embryonal cell line(293-EBNA) derived from kidney epithelial tissue transfected with bgal.The cells were transformed with adenovirus 5 DNA. BRACNOK02 PSPORT1 Thisamplified and normalized library was constructed using RNA isolated fromposterior cingulate tissue removed from an 85-year-old Caucasian femalewho died from myocardial infarction and retroperitoneal hemorrhage.Pathology indicated atherosclerosis, moderate to severe, involving thecircle of Willis, middle cerebral, basilar and vertebral arteries;infarction, remote, left dentate nucleus; and amyloid plaque depositionconsistent with age. There was mild to moderate leptomeningeal fibrosis,especially over the convexity of the frontal lobe. There was mildgeneralized atrophy involving all lobes. The white matter was mildlythinned. Cortical thickness in the temporal lobes, both maximal andminimal, was slightly reduced. The substantia nigra pars compactaappeared mildly depigmented. Patient history included COPD,hypertension, and recurrent deep venous thrombosis. 6.4 millionindependent clones from this amplified library were normalized in oneround using conditions adapted from Soares et al., PNAS (1994) 91:9228-9232 and Bonaldo et al., Genome Research 6 (1996): 791. BRAENOT02pINCY Library was constructed using RNA isolated from posterior parietalcortex tissue removed from the brain of a 35-year-old Caucasian male whodied from cardiac failure. BRAIFEE05 PCDNA2.1 This 5′ biased randomprimed library was constructed using RNA isolated from brain tissueremoved from a Caucasian male fetus who was stillborn with a hypoplasticleft heart at 23 weeks' gestation. BRAIFEN03 pINCY This normalized fetalbrain tissue library was constructed from 3.26 million independentclones from a fetal brain library. Starting RNA was made from braintissue removed from a Caucasian male fetus, who was stillborn with ahypoplastic left heart at 23 weeks' gestation. The library wasnormalized in 2 rounds using conditions adapted from Soares et al., PNAS(1994) 91: 9228 and Bonaldo et al., Genome Research (1996) 6: 791,except that a significantly longer (48 hours/round) reannealinghybridization was used. BRAIFER06 PCDNA2.1 This random primed librarywas constructed using RNA isolated from brain tissue removed from aCaucasian male fetus who was stillborn with a hypoplastic left heart at23 weeks' gestation. Serologies were negative. BRAITUT12 pINCY Librarywas constructed using RNA isolated from brain tumor tissue removed fromthe left frontal lobe of a 40-year-old Caucasian female during excisionof a cerebral meningeal lesion. Pathology indicated grade 4 gemistocyticastrocytoma. BRAIUNT01 pINCY Library was constructed using RNA isolatedfrom SK-N-MC, a neuroepithelioma cell line (ATCC HTB-10) derived from a14-year-old Caucasian female with neuroepithelioma, with metastasis tothe supra-orbital area. BRANDIN01 pINCY This normalized pineal glandtissue library was constructed from .4 million independent clones from apineal gland tissue library from two different donors. Starting RNA wasmade from pooled pineal gland tissue removed from two Caucasian females:a 68-year-old (donor A) who died from congestive heart failure and a79-year old (donor B) who died from pneumonia. Neuropathology for donorA indicated mild to moderate Alzheimer disease, atherosclerosis, andmultiple infarctions. Neuropathology for donor B indicated severeAlzheimer disease, arteriolosclerosis, cerebral amyloid angiopathy andmultiple infarctions. There were diffuse and neuritic amyloid plaquesand neurofibrillary tangles throughout the brain sections examined inboth donors. Patient history included diabetes mellitus, rheumatoidarthritis, hyperthyroidism, amyloid heart disease, and dementia in donorA; and pseudophakia, gastritis with bleeding, glaucoma, peripheralvascular disease, COPD, delayed onset tonic/clonic seizures, andtransient ischemic attack in donor B. The library was normalized in oneround using conditions adapted from Soares et al., PNAS (1994) 91:9228-9232 and Bonaldo et al., Genome Research 6 (1996): 791, except thata significantly longer (48 hours/round) reannealing hybridization wasused. BRAUNOR01 pINCY This random primed library was constructed usingRNA isolated from striatum, globus pallidus and posterior putamen tissueremoved from an 81-year-old Caucasian female who died from a hemorrhageand ruptured thoracic aorta due to atherosclerosis. Pathology indicatedmoderate atherosclerosis involving the internal carotids, bilaterally;microscopic infarcts of the frontal cortex and hippocampus, andscattered diffuse amyloid plaques and neurofibrillary tangles,consistent with age. Grossly, the leptomeninges showed only mildthickening and hyalinization along the superior sagittal sinus. Theremainder of the leptomeninges was thin and contained some congestedblood vessels. Mild atrophy was found mostly in the frontal poles andlobes, and temporal lobes, bilaterally. Microscopically, there werepairs of Alzheimer type II astrocytes within the deep layers of theneocortex. There was increased satellitosis around neurons in the deepgray matter in the middle frontal cortex. The amygdala contained rarediffuse plaques and neurofibrillary tangles. The posterior hippocampuscontained a microscopic area of cystic cavitation with hemosiderin-ladenmacrophages surrounded by reactive gliosis. Patient history includedsepsis, cholangitis, post-operative atelectasis, pneumonia CAD,cardiomegaly due to left ventricular hypertrophy, splenomegaly,arteriolonephrosclerosis, nodular colloidal goiter, emphysema, CHF,hypothyroidism, and peripheral vascular disease. CERVNOT01 PSPORT1Library was constructed using RNA isolated from the uterine cervicaltissue of a 35-year-old Caucasian female during a vaginal hysterectomywith dilation and curettage. Pathology indicated mild chroniccervicitis. Family history included atherosclerotic coronary arterydisease and type II diabetes. COLENOR03 PCDNA2.1 Library was constructedusing RNA isolated from colon epithelium tissue removed from a13-year-old Caucasian female who died from a motor vehicle accident.COLNFET02 pINCY Library was constructed using RNA isolated from thecolon tissue of a Caucasian female fetus, who died at 20 weeks'gestation. COLNPOT01 pINCY Library was constructed using RNA isolatedfrom colon polyp tissue removed from a 40-year-old Caucasian femaleduring a total colectomy. Pathology indicated an inflammatorypseudopolyp; this tissue was associated with a focally invasive grade 2adenocarcinoma and multiple tubuvillous adenomas. Patient historyincluded a benign neoplasm of the bowel. CONNNOT01 pINCY Library wasconstructed using RNA isolated from mesentery fat tissue obtained from a71-year-old Caucasian male during a partial colectomy and permanentcolostomy. Family history included atherosclerotic coronary arterydisease, myocardial infarction, and extrinsic asthma. CONTTUT01 pINCYLibrary was constructed using RNA isolated from tumorous soft tissue ofthe left lateral thigh removed from a 34-year-old Caucasian femaleduring a soft tissue excision. Pathology indicated metastatic grade 2myxoid liposarcoma which formed multiple, lobulated, circumscribedmasses situated in the subcutaneous adipose tissue. Patient historyincluded a malignant soft tissue neoplasm of the leg. Family historyincluded benign hypertension, acute leukemia, benign hypertension, andtype II diabetes. ENDCNOT03 pINCY Library was constructed using RNAisolated from dermal microvascular endothelial cells removed from aneonatal Caucasian male. EPIPNOT01 pINCY Library was constructed usingRNA isolated from prostatic epithelial cells removed from a 17-year-oldHispanic male. FIBRTXS07 pINCY This subtracted library was constructedusing 1.3 million clones from a dermal fibroblast library and wassubjected to two rounds of subtraction hybridization with 2.8 millionclones from an untreated dermal fibroblast tissue library. The startinglibrary for subtraction was constructed using RNA isolated from treateddermal fibroblast tissue removed from the breast of a 31-year-oldCaucasian female. The cells were treated with 9CIS retinoic acid. Thehybridization probe for subtraction was derived from a similarlyconstructed library from RNA isolated from untreated dermal fibroblasttissue from the same donor. Subtractive hybridization conditions werebased on the methodologies of Swaroop et al., NAR (1991) 19: 1954 andBonaldo, et al., Genome Research (1996) 6: 791. FIBRUNT02 pINCY Librarywas constructed using RNA isolated from an untreated MG-63 cell linederived from an osteosarcoma removed from a 14-year-old Caucasian male.GBLATUT01 pINCY Library was constructed using RNA isolated fromgallbladder tumor tissue removed from a 78-year-old Caucasian femaleduring a cholecystectomy. Pathology indicated invasive grade 2 squamouscell carcinoma, forming a mass in the gallbladder. Patient historyincluded diverticulitis of the colon, palpitations, benign hypertension,and hyperlipidemia. Family history included a cholecystectomy,atherosclerotic coronary artery disease, atherosclerotic coronary arterydisease, hyperlipidemia, and benign hypertension. HEAONOE01 PCDNA2.1This 5′ biased random primed library was constructed using RNA isolatedfrom the aorta of a 39-year-old Caucasian male, who died from a gunshotwound. Serology was positive for cytomegalovirus (CMV). Patient historyincluded tobacco abuse (one pack of cigarettes per day for 25 years),and occasionally cocaine, marijuana, and alcohol use. HEARNON03 pINCYThis normalized heart tissue library was constructed from 8.4 millionindependent clones from a heart tissue library. Starting RNA was madefrom heart tissue removed from a 44-year-old Caucasian male, who diedfrom intracranial hemorrhage. Serology was positive for anti-CMV(cytomegalovirus). Patient history included back and neck pain,hypertension, pneumonia, sinus infection, alcohol use, and daily pipetobacco use (×3 years). Patient medications included Procardia. Thelibrary was normalized in two rounds using conditions adapted fromSoares et al., PNAS (1994) 91: 9228-9232 and Bonaldo et al., GenomeResearch (1996) 6: 791, except that a significantly longer (48hours/round) reannealing hybridization was used. HNT2RAT01 PBLUESCRIPTLibrary was constructed at Stratagene (STR937231), using RNA isolatedfrom the hNT2 cell line (derived from a human teratocarcinoma thatexhibited properties characteristic of a committed neuronal precursor).Cells were treated with retinoic acid for 24 hours. KERANOT01PBLUESCRIPT Library was constructed using RNA isolated from neonatalkeratinocytes obtained from the leg skin of a spontaneously abortedblack male. KIDEUNE02 pINCY This 5′ biased random primed library wasconstructed using RNA isolated from an untreated transformed embryonalcell line (293-EBNA) derived from kidney epithelial tissue (Invitrogen).The cells were transformed with adenovirus 5 DNA. LUNGDIS03 pINCYLibrary was constructed using diseased lung tissue. 0.76 million clonesfrom a diseased lung tissue library were subjected to two rounds ofsubtraction hybridization with 5.1 million clones from a normal lungtissue library. The starting library for subtraction was constructedusing polyA RNA isolated from diseased lung tissue. Patient historyincluded idiopathic pulmonary disease. Subtractive hybridizationconditions were based on the methodologies of Swaroop et al. (1991)Nucleic Acids Res. 19: 1954; and Bonaldo et al. Genome Res. (1996) 6:791. LUNGTUT17 pINCY Library was constructed using RNA isolated fromlung tumor tissue removed from a 53-year-old male. Pathology indicatedgrade 4 adenocarcinoma. LUNLTUT11 pINCY Library was constructed usingRNA isolated from lung tumor tissue removed from the right upper lobe ofa 50-year-old Caucasian male during segmental lung resection. Pathologyindicated an invasive grade 4 squamous cell adenocarcinoma forming asubpleural mass, which puckered the underlying pleura. The tumor did notinfiltrate the pleura. Reactive mesothelial cells and fibrin werepresent at the right lower lobe of pleural implant. Patient historyincluded a respiratory anomaly, chest pain, and tobacco abuse. Familyhistory included skin cancer and type II diabetes. MCLDTXN05 pINCY Thisnormalized dendritic cell library was constructed from 1 millionindependent clones from a pool of two derived dendritic cell libraries.Starting libraries were constructed using RNA isolated from untreatedand treated derived dendritic cells from umbilical cord blood CD34+precursor cells removed from a male. The cells were derived withgranulocyte/macrophage colony stimulating factor (GM-CSF), tumornecrosis factor alpha (TNF alpha), and stem cell factor (SCF). TheGM-CSF was added at time 0 at 100 ng/ml the TNF alpha was added at time0 at 2.5 ng/ml, and the SCF was added at time 0 at 25 ng/ml. Incubationtime was 13 days. The treated cells were then exposed to phorbolmyristate acetate (PMA), and Ionomycin. The PMA and Ionomycin were addedat 13 days for five hours. The library was normalized in two roundsusing conditions adapted from Soares et al., PNAS (1994) 91: 9228 andBonaldo et al., Genome Research 6 (1996): 791, except that asignificantly longer (48 hours/round) reannealing hybridization wasused. MMLR3DT01 PSPORT1 Library was constructed using RNA isolated fromadherent mononuclear cells, which came from a pool of male and femaledonors. MUSCNOT10 pINCY Library was constructed using RNA isolated fromgluteal muscle tissue removed from a 43-year-old Caucasian female duringsoft tissue excision, partial ostectomy, and plastic skin repair.Pathology for the associated tumor tissue indicated recurrent clear cellsarcoma of soft parts, forming a mass in the coccygeal region,associated with a cystic cavity (previous biopsy site). Family historyincluded benign hypertension, osteoarthritis, prostate cancer,depression, osteoarthritis, benign hypertension, colon cancer, anddepression. NERDTDN03 pINCY This normalized dorsal root ganglion tissuelibrary was constructed from 1.05 million independent clones from adorsal root ganglion tissue library. Starting RNA was made from dorsalroot ganglion tissue removed from the cervical spine of a 32-year-oldCaucasian male who died from acute pulmonary edema, acutebronchopneumonia, bilateral pleural effusions, pericardial effusion, andmalignant lymphoma (natural killer cell type). The patient presentedwith pyrexia of unknown origin, malaise, fatigue, and gastrointestinalbleeding. Patient history included probable cytomegalovirus infection,liver congestion, and steatosis, splenomegaly, hemorrhagic cystitis,thyroid hemorrhage, respiratory failure, pneumonia of the left lung,natural killer cell lymphoma of the pharynx, Bell's palsy, and tobaccoand alcohol abuse. Previous surgeries included colonoscopy, closed colonbiopsy, adenotonsillectomy, and nasopharyngeal endoscopy and biopsy.Patient medications included Diflucan (fluconazole), Deltasone(prednisone), hydrocodone, Lortab, Alprazolam, Reazodone,ProMace-Cytabom, Etoposide, Cisplatin, Cytarabine, and dexamethasone.The patient received radiation therapy and multiple blood transfusions.The library was normalized in 2 rounds using conditions adapted fromSoares et al., PNAS (1994) 91: 9228-9232 and Bonaldo et al., GenomeResearch 6 (1996): 791, except that a significantly longer (48hours/round) reannealing hybridization was used. NOSEDIC02 PSPORT1 Thislarge size fractionated library was constructed using RNA isolated fromnasal polyp tissue. OVARDIJ01 pIGEN This random primed 5′ cap isolatedlibrary was constructed using RNA isolated from diseased right ovarytissue removed from a 47-year-old Caucasian female during totalabdominal hysterectomy, dilation and curettage, bilateralsalpingo-oophorectomy, repair of ureter, and incidental appendectomy.Pathology indicated endometriosis. Pathology for the associated tumortissue indicated multiple leiomyomata. The left ovary contained a corpusluteum. There was endometriosis involving the posterior serosa. Thepatient presented with metrorrhagia and a benign neoplasm of the ovary.Patient history included normal delivery, joint pain in multiple joints,and unilateral congenital hip dislocation. Previous surgeries includedtotal hip replacement. Patient medications included calcium. Familyhistory included kidney cancer in the mother; atherosclerotic coronaryartery disease and aortocoronary bypass of 3 coronary arteries in thefather; benign hypertension and Hodgkin's disease in the sibling(s); andbenign hypertension and cerebrovascular accident in the grandparent(s).PANCDIR02 PCDNA2.1 This random primed library was constructed using RNAisolated from diseased pancreatic tissue removed from a 43-year-oldCaucasian female who died from a gunshot wound to the head. Patienthistory included type I diabetes for 38 years, a fractured finger, andtobacco use (1 pack per day for 25 years). The serology was positive CMVantibody and remaining serologies were negative. Patient medicationsincluded antidepressants and Insulin. PANCNOE02 PCDNA2.1 This 5′ biasedrandom primed library was constructed using RNA isolated from pancreatictissue removed from an 8-year-old Black male, who died from anoxia.Serologies were negative. Patient medications included DDAVP, Versed,and labetalol. PANCNOT04 PSPORT1 Library was constructed using RNAisolated from the pancreatic tissue of a 5-year-old Caucasian male whodied in a motor vehicle accident. PANCNOT05 PSPORT1 Library wasconstructed using RNA isolated from the pancreatic tissue of a2-year-old Hispanic male who died from cerebral anoxia. PANCTUT02 pINCYLibrary was constructed using RNA isolated from pancreatic tumor tissueremoved from a 45-year-old Caucasian female during radicalpancreaticoduodenectomy. Pathology indicated a grade 4 anaplasticcarcinoma. Family history included benign hypertension, hyperlipidemiaand atherosclerotic coronary artery disease. PENITUT01 pINCY Library wasconstructed using RNA isolated from tumor tissue removed from the penisof a 64-year-old Caucasian male during penile amputation. Pathologyindicated a fungating invasive grade 4 squamous cell carcinoma involvingthe inner wall of the foreskin and extending onto the glans penis.Patient history included benign neoplasm of the large bowel,atherosclerotic coronary artery disease, angina pectoris, gout, andobesity. Family history included malignant pharyngeal neoplasm, chroniclymphocytic leukemia, and chronic liver disease. PROSBPT07 pINCY Librarywas constructed using RNA isolated from diseased prostate tissue removedfrom a 53-year-old Caucasian male during radical prostatectomy andregional lymph node excision. Pathology indicated adenofibromatoushyperplasia. Pathology for the associated tumor tissue indicatedadenocarcinoma (Gleason grade 3 + 2). The patient presented withelevated prostate specific antigen and induration. Patient historyincluded hyperlipidemia. Family history included atheroscleroticcoronary artery disease, coronary artery bypass graft, perforatedgallbladder, hyperlipidemia, and kidney stones. PROSTME06 PCDNA2.1 This5′ biased random primed library was constructed using RNA isolated fromdiseased prostate tissue removed from a 57-year-old Caucasian maleduring closed prostatic biopsy, radical prostatectomy, and regionallymph node excision. Pathology indicated adenofibromatous hyperplasia.Pathology for the matched tumor tissue indicated adenocarcinoma, Gleasongrade 3 + 3, forming a predominant mass involving the right sidecentrally. The patient presented with elevated prostate specific antigenand prostate cancer. Patient history included tobacco abuse inremission. Previous surgeries included cholecystectomy, repair ofdiaphragm hernia, and repair of vertebral fracture. Patient medicationsincluded Pepsid, Omnipen, and Eulexin. Family history included benignhypertension, cerebrovascular accident, atherosclerotic coronary arterydisease, uterine cancer and type II diabetes in the mother; prostatecancer in the father; drug abuse, prostate cancer, and breast cancer inthe sibling(s). SCORNON02 PSPORT1 This normalized spinal cord librarywas constructed from 3.24M independent clones from the a spinal cordtissue library. RNA was isolated from the spinal cord tissue removedfrom a 71-year-old Caucasian male who died from respiratory arrest.Patient history included myocardial infarction, gangrene, and end stagerenal disease. The normalization and hybridization conditions wereadapted from Soares et al. (PNAS (1994) 91: 9228). SCORNOT04 pINCYLibrary was constructed using RNA isolated from cervical spinal cordtissue removed from a 32-year-old Caucasian male who died from acutepulmonary edema and bronchopneumonia, bilateral pleural and pericardialeffusions, and malignant lymphoma (natural killer cell type). Patienthistory included probable cytomegalovirus infection, hepatic congestionand steatosis, splenomegaly, hemorrhagic cystitis, thyroid hemorrhage,and Bell's palsy. Surgeries included colonoscopy, large intestinebiopsy, adenotonsillectomy, and nasopharyngeal endoscopy and biopsy;treatment included radiation therapy. TESTNON04 pINCY This normalizedtestis tissue library was constructed from 6.48 million independentclones from a pool of testis tissue libraries. Starting RNA was madefrom testicular tissue removed from a 16-year-old Caucasian male whodied from hanging. The library was normalized in two rounds usingconditions adapted from Soares et al., PNAS (1994) 91: 9228 and Bonaldoet al., Genome Research 6 (1996): 791, except that a significantlylonger (48-hours/round)reannealing hybridization was used. TESTTUT02pINCY Library was constructed using RNA isolated from testicular tumorremoved from a 31-year-old Caucasian male during unilateral orchiectomy.Pathology indicated embryonal carcinoma. THP1NOT03 pINCY Library wasconstructed using RNA isolated from untreated THP-1 cells. THP-1 is ahuman promonocyte line derived from the peripheral blood of a 1-year-oldCaucasian male with acute monocytic leukemia (ref: Int. J. Cancer (1980)26: 171). THYMNOE02 PCDNA2.1 This 5′ biased random primed library wasconstructed using RNA isolated from thymus tissue removed from a3-year-old Hispanic male during a thymectomy and closure of a patentductus arteriosus. The patient presented with severe pulmonary stenosisand cyanosis. Patient history included a cardiac catheterization andechocardiogram. Previous surgeries included Blalock-Taussig shunt andpulmonary valvotomy. The patient was not taking any medications. Familyhistory included benign hypertension, osteoarthritis, depressivedisorder, and extrinsic asthma in the grandparent(s). THYRDIE01 PCDNA2.1This 5′ biased random primed library was constructed using RNA isolatedfrom diseased thyroid tissue removed from a 22-year-old Caucasian femaleduring closed thyroid biopsy, partial thyroidectomy, and regional lymphnode excision. Pathology indicated adenomatous hyperplasia. The patientpresented with malignant neoplasm of the thyroid. Patient historyincluded normal delivery, alcohol abuse, and tobacco abuse. Previoussurgeries included myringotomy. Patient medications included anunspecified type of birth control pills. Family history includedhyperlipidemia and depressive disorder in the mother; and benignhypertension, congestive heart failure, and chronic leukemia in thegrandparent(s). UTRCDIE01 PCDNA2.1 This 5′ biased random primed librarywas constructed using RNA isolated from uterine cervix tissue removedfrom a 29-year-old Caucasian female during a vaginal hysterectomy andcystocele repair. Pathology indicated the cervix showed mild chroniccervicitis with focal squamous metaplasia. Pathology for the matchedtumor tissue indicated intramural uterine leiomyoma. Patient historyincluded hypothyroidism, pelvic floor relaxation, paraplegia, and selfcatheterization. Previous surgeries included a normal delivery, alaminectomy, and a rhinoplasty. Patient medications included Synthroid.Family history included benign hypertension in the father; and type IIdiabetes and hyperlipidemia in the mother. UTRSDIC01 PSPORT1 This largesize fractionated library was constructed using pooled cDNA from eightdonors. cDNA was generated using mRNA isolated from endometrial tissueremoved from a 32-year-old female (donor A); endometrial tissue removedfrom a 32-year-old Caucasian female (donor B) during abdominalhysterectomy, bilateral salpingo-oophorectomy, and cystocele repair;from diseased endometrium and myometrium tissue removed from a38-year-old Caucasian female (donor C) during abdominal hysterectomy,bilateral salpingo-oophorectomy, and exploratory laparotomy; fromendometrial tissue removed from a 41-year-old Caucasian female (donor D)during abdominal hysterectomy with removal of a solitary ovary; fromendometrial tissue removed from a 43-year-old Caucasian female (donor E)during vaginal hysterectomy, dilation and curettage, cystocele repair,rectocele repair and cystostomy; and from endometrial tissue removedfrom a 48-year-old Caucasian female (donor F) during a vaginalhysterectomy, rectocele repair, and bilateral salpingo-oophorectomy.Pathology (A) indicated the endometrium was in secretory phase.Pathology (B) indicated the endometrium was in the proliferative phase.Pathology (C) indicated extensive adenomatous hyperplasia with squamousmetaplasia and focal atypia, forming a polypoid mass within theendometrial cavity. The cervix showed chronic cervicitis and squamousmetaplasia. Pathology (D, E) indicated the endometrium was secretoryphase. Pathology (F) indicated the endometrium was weakly proliferative.UTRSNOT02 PSPORT1 Library was constructed using RNA isolated fromuterine tissue removed from a 34-year-old Caucasian female during avaginal hysterectomy. Patient history included mitral valve disorder.Family history included stomach cancer, congenital heart anomaly,irritable bowel syndrome, ulcerative colitis, colon cancer,cerebrovascular disease, type II diabetes, and depression. UTRSTMR01pINCY Library was constructed using RNA isolated from uterine myometrialtissue removed from a 41-year-old Caucasian female during a vaginalhysterectomy. The endometrium was secretory and contained fragments ofendometrial polyps. Pathology for associated tumor tissue indicateduterine leiomyoma. Patient history included ventral hernia and a benignovarian neoplasm.

TABLE 7 Program Description Reference Parameter Threshold ABI A programthat removes vector Applied Biosystems, Foster City, CA. FACTURAsequences and masks ambiguous bases in nucleic acid sequences. ABI/ AFast Data Finder useful in Applied Biosystems, Foster City, CA; Mismatch<50% PARACEL comparing and annotating amino Paracel Inc., Pasadena, CA.FDF acid or nucleic acid sequences. ABI A program that assembles AppliedBiosystems, Foster City, CA. AutoAssembler nucleic acid sequences. BLASTA Basic Local Alignment Search Altschul, S. F. et al. (1990) J. Mol.Biol. ESTs: Probaility value = 1.0E−8 Tool useful in sequence 215:403-410; Altschul, S. F. et al. (1997) or less similarity search foramino Nucleic Acids Res. 25: 3389-3402. Full Length sequences:Probability acid and nucleic acid sequences. value = 1.0E−10 or lessBLAST includes five functions: blastp, blastn, blastx, tblastn, andtblastx. FASTA A Pearson and Lipman Pearson, W. R. and D. J. Lipman(1988) Proc. ESTs: fasta E value = 1.06E−6 algorithm that searches forNatl. Acad Sci. USA 85: 2444-2448; Pearson, W. R. Assembled ESTs: fastaIdentity = 95% similarity between a query (1990) Methods Enzymol. 183:63-98; or greater and sequence and a group of and Smith, T. F. and M. S.Waterman (1981) Match length = 200 bases or greater; sequences of thesame type. Adv. Appl. Math. 2: 482-489. fastx E value = 1.0E−8 or lessFASTA comprises as least five Full Length sequences: functions: fasta,tfasta, fastx, fastx score = 100 or greater tfastx, and ssearch. BLIMPSA BLocks IMProved Searcher Henikoff, S. and J. G. Henikoff (1991)Nucleic Probability value = 1.0E−3 or less that matches a sequenceagainst Acids Res. 19: 6565-6572; Henikoff, J. G. & S. Henikoff those inBLOCKS, PRINTS, (1996) Methods Enzymol. 266: 88-105; DOMO, PRODOM, andPFAM and Attwood, T. K. et al. (1997) J. Chem. databases to search forgene Inf. Comput. Sci. 37: 417-424. families, sequence homology, andstructural fingerprint regions. HMMER An algorithm for searching aKrogh, A. et al. (1994) J. Mol. Biol. 235: 1501-1531; PFAM, INCY, SMART,or TIGRFAM query sequence against hidden Sonnhammer, E. L. L. et al.(1988) hits: Probability value = 1.0E−3 or less Markov model (HMM)-basedNucleic Acids Res. 26: 320-322; Durbin, R. et Signal peptide hits: Score= 0 or databases of protein family al. (1998) Our World View, in aNutshell, greater consensus sequences, such as Cambridge Univ. Press, p.1-350 PFAM, INCY, SMART, and TIGRFAM. ProfileScan An algorithm thatsearches Gribskov, M. et al. (1988) CABIOS 4: 61-66; Normalized qualityscore ≧ GCG- for structural and sequence Gribskov, M. et al. (1989)Methods Enzymol. specified “HIGH” value for that motifs in proteinsequences 183: 146-159; Bairoch, A. et al. (1997) particular Prositemotif. that match sequence patterns Nucleic Acids Res. 25: 217-221.Generally, score = 1.4-2.1. defined in Prosite. Phred A base-callingalgorithm that Ewing, B. et al. (1998) Genome Res. examines automatedsequencer 8: 175-185; Ewing, B. and P. Green traces with highsensitivity (1998) Genome Res. 8: 186-194. and probability. Phrap APhils Revised Assembly Smith, T. F. and M. S. Waterman (1981) Adv. Score= 120 or greater; Program including SWAT and Appl. Math. 2: 482-489;Smith, T. F. and M. S. Waterman Match length = 56 or greater CrossMatch,programs based (1981) J. Mol. Biol. 147: 195-197; on efficientimplementation and Green, P., University of Washington, of theSmith-Waterman Seattle, WA. algorithm, useful in searching sequencehomology and assembling DNA sequences. Consed A graphical tool forviewing and Gordon, D. et al. (1998) Genome Res. 8: 195-202. editingPhrap assemblies. SPScan A weight matrix analysis Nielson, H. et al.(1997) Protein Engineering Score = 3.5 or greater program that scansprotein 10: 1-6; Claverie, J. M. and S. Audic (1997) sequences for thepresence CABIOS 12: 431-439. of secretory signal peptides. TMAP Aprogram that uses weight Persson, B. and P. Argos (1994) J. Mol. Biol.matrices to delineate 237: 182-192; Persson, B. and P. Argos (1996)transmembrane segments on Protein Sci. 5: 363-371. protein sequences anddetermine orientation. TMHMMER A program that uses a hidden Sonnhammer,E. L. et al. (1998) Proc. Sixth Intl. Markov model (HMM) to Conf. onIntelligent Systems for Mol. Biol., delineate transmembrane Glasgow etal., eds., The Am. Assoc. for Artificial segments on protein sequencesIntelligence Press, Menlo Park, CA, pp. 175-182. and determineorientation. Motifs A program that searches amino Bairoch, A. et al.(1997) Nucleic Acids Res. 25: 217-221; acid sequences for patterns thatWisconsin Package Program Manual, version 9, page matched those definedin M51-59, Genetics Computer Group, Madison, WI. Prosite.

1. An isolated polypeptide selected from the group consisting of: a) apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-17, SEQ ID NO:23-25, and SEQ ID NO:28-58, b) apolypeptide consisting essentially of a naturally occurring amino acidsequence selected from the group consisting of SEQ ID NO: 18-22 and SEQID NO:26-27, c) a polypeptide consisting essentially of a naturallyoccurring amino acid sequence at least 90% identical to an amino acidsequence consisting of SEQ ID NO: 1, SEQ ID NO:3-5, SEQ ID NO:9, SEQ IDNO:15, SEQ ID NO:18-22, SEQ ID NO:26-27, SEQ ID NO:35-36, SEQ ID NO:41,SEQ ID NO:49-50, SEQ ID NO:53, and SEQ ID NO:58, d) a polypeptidecomprising a naturally occurring amino acid sequence at least 90%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO: 10-11, SEQ ID NO:13, SEQ IDNO:16, SEQ ID NO:28-29, SEQ ID NO:31-34, SEQ ID NO:39-40, SEQ IDNO:42-43, SEQ ID NO:46, SEQ ID NO:52, and SEQ ID NO:57, e) a polypeptidecomprising a naturally occurring amino acid sequence at least 91%identical to the amino acid sequence of SEQ ID NO:47, f) a polypeptidecomprising a naturally occurring amino acid sequence at least 92%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:23, and SEQ ID NO:38, g) a polypeptide comprising anaturally occurring amino acid sequence at least 93% identical to theamino acid sequence of SEQ ID NO:55, h) a polypeptide comprising anaturally occurring amino acid sequence at least 94% identical to theamino acid sequence of SEQ ID NO:24, i) a polypeptide comprising anaturally occurring amino acid sequence at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:12,SEQ ID NO: 14, SEQ ID NO:37, and SEQ ID NO:56, j) a polypeptidecomprising a naturally occurring amino acid sequence at least 96%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO: 17, and SEQ ID NO:48, k) a polypeptidecomprising a naturally occurring amino acid sequence at least 97%identical to an amino acid sequence selected from the group consistingof SEQ ID NO:30, and SEQ ID NO:45, l) a polypeptide comprising anaturally occurring amino acid sequence at least 99% identical to anamino acid sequence selected from the group consisting of SEQ ID NO:25,SEQ ID NO:44, SEQ ID NO:51, and SEQ ID NO:54, m) a biologically activefragment of a polypeptide having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 1-58, and n) an immunogenic fragmentof a polypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-58.
 2. An isolated polypeptide of claim 1selected from the group consisting of: a) a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1-17, SEQ ID NO 23-25, and SEQ ID NO:28-58, and b) a polypeptideconsisting essentially of an amino acid sequence selected from the groupconsisting of SEQ ID NO:18-22, and SEQ ID NO:26-27.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4 comprising a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:59-116.
 6. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 3. 7. A cell transformed with a recombinantpolynucleotide of claim
 6. 8. (CANCELED)
 9. A method of producing apolypeptide of claim 1, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 1, and b) recoveringthe polypeptide so expressed.
 10. A method of claim 9, wherein thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1-58.
 11. An isolated antibody whichspecifically binds to a polypeptide of claim
 1. 12. An isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:59-116, b) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:59-108 and SEQ ID NO:110-116, c) a polynucleotide comprising anaturally occurring polynucleotide sequence at least 97% identical tothe polynucleotide sequence of SEQ ID NO: 109, d) a polynucleotidecomplementary to a polynucleotide of a), e) a polynucleotidecomplementary to a polynucleotide of b), f) a polynucleotidecomplementary to a polynucleotide of c), and g) an RNA equivalent ofa)-f).
 13. (CANCELED)
 14. A method of detecting a target polynucleotidein a sample, said target polynucleotide having a sequence of apolynucleotide of claim 12, the method comprising: a) hybridizing thesample with a probe comprising at least 20 contiguous nucleotidescomprising a sequence complementary to said target polynucleotide in thesample, and which probe specifically hybridizes to said targetpolynucleotide, under conditions whereby a hybridization complex isformed between said probe and said target polynucleotide or fragmentsthereof, and b) detecting the presence or absence of said hybridizationcomplex, and, optionally, if present, the amount thereof.
 15. (CANCELED)16. A method of detecting a target polynucleotide in a sample, saidtarget polynucleotide having a sequence of a polynucleotide of claim 12,the method comprising: a) amplifying said target polynucleotide orfragment thereof using polymerase chain reaction amplification, and b)detecting the presence or absence of said amplified targetpolynucleotide or fragment thereof, and, optionally, if present, theamount thereof.
 17. A composition comprising a polypeptide of claim 1and a pharmaceutically acceptable excipient.
 18. A composition of claim17, wherein the polypeptide is selected from the group consisting of: a)a polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-17, SEQ ID NO:23-25, and SEQ ID NO:28-58, andb) a polypeptide consisting essentially of an amino acid sequenceselected from the group consisting of SEQ ID NO:18-22, and SEQ IDNO:26-27.
 19. (CANCELED)
 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample. 21.(CANCELED)
 22. (CANCELED)
 23. A method of screening a compound foreffectiveness as an antagonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting antagonist activity in the sample. 24.(CANCELED)
 25. (CANCELED)
 26. A method of screening for a compound thatspecifically binds to the polypeptide of claim 1, the method comprising:a) combining the polypeptide of claim 1 with at least one test compoundunder suitable conditions, and b) detecting binding of the polypeptideof claim 1 to the test compound, thereby identifying a compound thatspecifically binds to the polypeptide of claim
 1. 27. (CANCELED)
 28. Amethod of screening a compound for effectiveness in altering expressionof a target polynucleotide, wherein said target polynucleotide comprisesa sequence of claim 5, the method comprising: a) exposing a samplecomprising the target polynucleotide to a compound, under conditionssuitable for the expression of the target polynucleotide, b) detectingaltered expression of the target polynucleotide, and c) comparing theexpression of the target polynucleotide in the presence of varyingamounts of the compound and in the absence of the compound.
 29. A methodof assessing toxicity of a test compound, the method comprising: a)treating a biological sample containing nucleic acids with the testcompound, b) hybridizing the nucleic acids of the treated biologicalsample with a probe comprising at least 20 contiguous nucleotides of apolynucleotide of claim 12 under conditions whereby a specifichybridization complex is formed between said probe and a targetpolynucleotide in the biological sample, said target polynucleotidecomprising a polynucleotide sequence of a polynucleotide of claim 12 orfragment thereof, c) quantifying the amount of hybridization complex,and d) comparing the amount of hybridization complex in the treatedbiological sample with the amount of hybridization complex in anuntreated biological sample, wherein a difference in the amount ofhybridization complex in the treated biological sample is indicative oftoxicity of the test compound. 30-171. (canceled)